BRPI0708791A2 - medicines and proteins - Google Patents

Abstract

DRUGS AND PROTEINS. The present invention relates to the use of monomeric TGF- <225> s, or fragments or derivatives thereof, as medicaments. These drugs preferably comprise TGF- <225> 3, or fragments or derivatives thereof. The medications provided can be used to accelerate wounds and / or inhibit scarring, to promote epithelial regeneration, or to prevent and / or treat fibrotic disorders.

Description

Report of the Invention Patent for "MEDICATIONS AND PROTEINS".

The present invention relates to the supply of new drugs. In preferred embodiments the invention provides medicaments suitable for use in accelerating wound healing and / or scar prevention, reduction or inhibition. The invention also provides a method for accelerating wound healing and / or for preventing, reducing or inhibiting scarring.

Transforming Growth Factor-Betas (TGF-Bs) is a family of cytokines having various biological activities. The TGF-β family comprises five isoforms, TGF-B1, TGF-B2, TGF-B3, TGF-B4, and TGF-B5.

TGF-Bs have utility in many different therapeutic contexts. As a result of the therapeutic potential of TGF-Bs has long been of interest in the pharmaceutical application of TGF-B family members, particularly TGF-Bs 1-3.

TGF-B1, TGF-B2 and TGF-B3 are all known to play crucial roles in regulating wound healing response. TGF-B activity can influence the wound healing rate as well as the extent of the scarce that occurs as a result of healing.

TGF-B1 has uses for the prevention and / or treatment of sclero-derma, angiogenesis disorders, kidney disease, osteoporosis, bone disease, glomerulonephritis and kidney disease.

TGF-B2 can be used to treat glioma, non-small cell lung cancer, pancreatic tumor, solid tumors, colon tumor, ovarian tumor, age-related macular degeneration, ocular injury, osteoporosis, retinopathy, ulcers, carcinoma, mouth inflammation and scleroderma.

TGF-B3 can be used to treat fibrotic disorders, pulmonary fi brosis, liver cirrhosis, scleroderma, angiogenesis disorders, restenosis, adhesions, endometriosis, ischemic disease, in vitropor fertilization of bone and cartilage, oral mucositis, kidney disease. , prevention, reduction or inhibition of scarring, improvement of neuronal reconnection in the peripheral and central nervous system, prevention, reduction or inhibition of complications from eye surgery (such as LASIK or PRK surgery) or scar in the back of the eye (such as associated scarring). proliferative vitreoretinopathy).

Members of the TGF-β family naturally exist as dimers comprising two peptide chains. TGF-Bacterial dimers have a molecular weight of approximately 25.4kDa. The active TGF-β fragmentodimeric is stabilized by hydrophobic and ionic interactions, which are further enhanced by an inter-subunit disulfide bridge. It is generally accepted that the biological activity, and thus all therapeutic activity, of members of the TGF-B family is extracted solely by the active dimer.

In the light of the foregoing, it will be recognized that there is a well-established need to provide medicines capable of utilizing the therapeutic properties of members of the TGF-B family. However, despite this well-recognized requirement for pharmaceutical compositions comprising members of the TGF-B family, there are many confirmed problems associated with the manufacture of TGF-Bs for therapeutic use.

One of the major disadvantages of using TGF-Bs as a therapeutic agent is that in order to biologically produce (and then therapeutically) active protein dimers using a prokaryotic host, intensive arenaturation to the biologically active dimeric form is required. Dimer formation can significantly extend the manufacturing time lines. This also has important implications for limiting the therapeutic use of any TGF-B.

It is an object of the present invention to prevent or mitigate at least some of the problems associated with prior art pharmaceutical compositions comprising TGF-Bs. In particular, an object of certain embodiments of the present invention is to prevent or mitigate problems associated with the use of time intensive manufacturing approaches used to produce biologically active dimers of these proteins.

In a first aspect of the invention there is provided a TGF-β monomer, or biologically active fragment thereof, for use as a medicament.

The invention is based on the very surprising discoveries of the inventors in which monomeric TGF-Bs may exert biological activities that were previously believed to be provided only by dimeric TGF-β proteins. These findings make it possible to use monomeric TGF-Bs in effective drugs capable of utilizing the biological properties of a selected TGF-B3. As will be readily appreciated in this finding and, therefore, the present invention, large expansions in practical applications where TGF-Bs can be therapeutically used, through the reduction of time and cost associated with the manufacture of suitable medicaments.

The inventors believe that the advantages of medicaments according to the invention may be provided by medicaments comprising any monomeric TGF-B isoform. Accordingly, except where the context otherwise requires, references to TGF-B or TGF-Bs may be taken to encompass any biologically active monomeric form of a TGF-B isoform such as TGF-B1, 2, 3, 4 and 5. Preferred monomers suitable for use in the medicaments of the invention are wild type TGF-B monomers (of any desired isoform).

Preferably monomeric TGF-Bs suitable for use according to the present invention are selected from the group consisting of TGF-B1, TGF-B2 and TGF-B3. More preferably the monomeric TGF-B is TGF-B3, and in a second aspect of the invention there is provided a TGF-B3 monomer, or a biologically active fragment thereof, for use as a medicament. It is preferred that the TGF-B monomers to be used in accordance with the present invention are human TGF-B monomers. It is preferred that TGF-Bs supplied in medicaments according to the present invention should be present substantially in monomeric form entirely. Monomeric TGF-Bs suitable for use in the medicaments or methods of the invention may preferably have a molecular weight of α-approximately 12kDa, and more particularly approximately 12.5kDa.

The amino acid residue sequences of the human forms of the isoforms TGF-βF, TGF-B2 and TGF-B3 are shown as Sequence ID Ne 1, 2 and 3 respectively, and a monomeric TGF-β comprising either of these sequences, or a biologically active fragment of any of these. sequences is a preferred monomer for use in accordance with the present invention. Monomeric TGF-B3 (Sequence ID No. 3) is a particularly preferred monomer for use in accordance with the various aspects of the present invention.

Except where the context otherwise requires, references in this report to monomeric TGF-Bs (including references to particular isoforms) should also be taken to encompass biologically active fragments and derivatives of such monomeric TGF-Bs. Sequence ID Nos. 1-3 represent preferred fragments for use in the medicaments of the invention, with biologically active fragments derived from Sequence ID No. 3 constituting particularly preferred fragments. Preferred biologically active fragments may comprise the receptor binding moiety of the selected TGF-β isoform.

Suitable derivatives of monomeric TGF-Bs that may be used in the medicaments or methods of the invention include: therapeutically effective peptide derivatives of monomeric TGF-Bs (or fragments thereof); therapeutically effective fragments or derivatives comprising or based on the pharmacophore of monomeric TGF-Bs (or fragments thereof); therapeutically effective peptide derivatives of monomeric TGF-Bs (or fragments thereof); therapeutically effective D-amino acid derivatives of monomeric TGF-Bs (or fragments thereof); therapeutically effective peptidomimetics based on monomeric TGF-Bs (or fragments thereof); therapeutically effective peptide analogs of TGF-Bsmonomeric (or fragments thereof); therapeutically effective pseudopeptides based on monomeric TGF-Bs (or fragments thereof); therapeutically effective retro-inverse peptides based on monomeric TGF-Bs (or fragments thereof); therapeutically effective depsipeptide derivatives based on monomeric TGF-Bs (or fragments thereof); therapeutically effective β-peptide derivatives based on monomeric TGF-Bs (or fragments thereof); and therapeutically effective retropeptide derivatives based on monomeric TGF-Bs (or fragments thereof).

The inventors have found that monomeric forms of human TGF-B3 can be used in the same manner as dimeric TGF-B3, for example for the treatment of fibrotic disorders, scleroderma, angiogenesis disorders, restenosis, adhesions, endometriosis, ischemic disease, bone and cartilage induction. , in vitro fertilization, oral mucositis, disease, prevention, scar reduction or inhibition, improvement of neuronal reconnection in the peripheral and central nervous system, prevention, reduction or inhibition of eye surgery complications (such as LASIK or PRK surgery), treats cleft palate and lip treatment, prevention, reduction or inhibition of healing accelerated healing of tendons and ligaments. Monomeric forms of TGF-B3 are also capable of promoting epithelial regeneration at sites of epithelial damage.

The inventors' findings also indicate that monomeric forms of TGF-B1 and TGF-B2 are capable of exerting all the therapeutic activities associated with dimeric forms of these growth factors.

"Biological activity" in the context of the present invention is preferably measured in vivo. The inventors have found that, according to findings published in the prior art, monomeric forms of TGF-Bs generally do not exhibit biological activity as assessed by invitro methods. However, in direct contrast to prior art beliefs, the inventors have found very surprisingly that monomeric forms of TGF-Bs can exert biological and therapeutic activity in vivo. Accordingly, it will be recognized that for a monomeric TGF-B, or a fragment or derivative thereof, to be deemed biologically active in accordance with the present invention, it is not necessary for the monomeric TGF-B to exhibit measurable biological activity. both media in vitro and in vivo, but merely exhibiting biological activity that can be measured in vitro or in vivo, and preferably activity that can be measured in vivo.

Suitably, the biological activity of monomeric TGF-Bs suitable for use according to the first aspect of the invention may be measured with reference to the known biological activity of TGF-β dated dimers. In the preferred embodiment, biological activity can be measured with reference to the ability of a monomer (such as a Sequence ID N-3 monomer) to accelerate wound healing and / or to prevent, reduce or inhibit scarring.

In view of the preceding paragraphs, it will be appreciated that medicaments according to the first or second aspect of the invention are particularly suitable for use in accelerating wound healing or scar prevention, reduction or inhibition. Furthermore, medicaments according to the first or second aspect of the invention are also particularly suitable for use in promoting reepithelialization.

Of course, in a third aspect of the invention there is provided the use of a TGF-B3 monomer, or a biologically active fragment thereof, in the preparation of a medicament for use in accelerating wound healing and / or scar inhibition.

In a fourth aspect of the invention there is provided the use of a TGF-B3 monomer, or biologically active fragment thereof, in the preparation of a medicament for use in promoting epithelial regeneration.

In a fifth aspect of the invention there is provided the use of a TGF-B3 monomer, or a biologically active fragment thereof in the preparation of a medicament for use in the prevention and / or treatment of a fibrotic disorder. Preferred fibrotic disorders that can be prevented and / or treated using such medicaments may be selected from the group consisting of lung fibrosis, liver fibrosis, sclero-derma, skin fibrosis, muscle fibrosis, fibrosis fibrosis. radiation, derim fibrosis and uterine fibrosis.

Furthermore, in a sixth aspect of the invention there is provided a method of accelerating wound healing and / or scar inhibition, the method comprising administering to a patient in need of such treatment a therapeutically effective amount of a monomeric TGF-B3, or fragment thereof.

In a seventh aspect of the invention there is provided an epithelial regeneration enhancement method, the method comprising administering to a patient in need of such treatment a therapeutically effective amount of a monomeric TGF-B3, or a fragment thereof.

In an eighth aspect of the invention there is provided a method of preventing and / or treating a fibrotic disorder, the method comprising administering to a patient in need of such prevention and / or treatment a therapeutically effective amount of a monomeric TGF-33. , or a fragment thereof. Preferred fibrotic disorders which may be prevented and / or treated using such methods may be selected from the group consisting of lung fibrosis, liver fibrosis, sclero-derma, skin fibrosis, muscle fibrosis, radiation fibrosis, derim fibrosis and fibrosis. uterine

For purposes of the present invention a therapeutically effective amount of a monomeric TGF-β, or a fragment thereof, is an amount sufficient to cause a required:

i) acceleration of wound healing and / or scar inhibition; or

ii) promotion of epithelial regeneration; or

iii) prevention and / or treatment of a fibrotic disorder.

The extent of acceleration of wound healing and / or inhibition of cation, or epithelial regeneration, or prevention or reduction of fibrosis that may be required will be apparent, and can certainly be readily determined by a attending physician. patient care. An appropriate assessment of the extent of acceleration of wound healing and / or scarred inhibition, or promotion of epithelial regeneration, or prevention or reduction of fibrosis may be determined by the clinician, and may be with reference to the measurement methods. suggested options described here.

Suitable and preferred monomeric TGF-Bs for use according to the invention may be selected with reference to any of the considerations described herein.

In the case where acceleration of wound healing is desired with scar prevention, reduction or inhibition, or prevention of other fibrotic disorders, for example pulmonary fibrosis, cirrhosis of the liver, scleroderma, glomerulonephritis, or the like, it is generally preferred to use Monomeric TGF-B3 or a suitable biologically active fragment.

Although the use of monomeric TGF-33 has particularly noteworthy benefits, drugs comprising monomeric forms of other TGF-β isoforms are also of great value.

For example, medicaments comprising monomeric TGF-BI, or biologically active fragments thereof, may have uses for the prevention and / or treatment of non-chronic wound healing, for example venous ulcers, pressure symptoms, diabetic ulcers, disorders of angiogenesis, kidney disease, osteoporosis, bone disease, glomerulonephritis and renal disease. As an example, distributed wild-type dimeric TGF-31 has been shown in animal models to be a powerful stimulator of bone growth and regeneration. In addition, the administration of TGF-βΙ has been shown to protect tissues from ischemic reperfusion disorder, particularly in the brain (followed by stroke), and in the heart (followed by occlusion of the coronary artery).

Medicines comprising monomeric TGF-B2, or biologically active fragments thereof, may be used in the treatment of glioma, non-small cell lung cancer, pancreatic tumor, solid tumors, colon tumor, ovarian tumor, macular degeneration. related to age, eye injury, osteoporosis, retinopathy, ulcers, carcinoma, mouth inflammation and scleroderma. As an example, topical application of wild-type dimeric TGF-32 has been shown in two clinical trials to accelerate closure of chronic leg ulcers associated with venous stasis; and local distribution of recombinant human TGF-332 in animal models has been shown to promote bone regrowth and regeneration. Medicines comprising monomeric TGF-B3, or biologically active fragments thereof, may be used in the treatment of wounds (including chronic wounds such as ulcers). ), scleroderma, fibrotic disorders, eg pulmonary fibrosis, liver cirrhosis, kidney fibrosis, etc., angiogenesis disorders, restenosis, adhesions, endometriosis, ischemic disease, bone and cartilage induction, in vitro fertilization, oral mucositis and kidney disease. As an example, topical application of wild-type dimeric TGF-B3 has been shown, in animal and clinical models, to accelerate the cure rate of chronic, non-healing pressure ulcers; reduce the incidence, severity, and duration of oral mucositis; and reduce the adverse side effects of gastrointestinal radiation syndrome resulting from damage to stem cells caused by radiotherapy and chemotherapy during cancer treatment. The inventors' findings indicate that monomeric TGF-β3 can be used in all of these contexts in order to cause known therapeutic activities of dimeric TGF-β.

The ability of certain inventive methods and drugs (such as those using TGF-β monomers as prepared in Sequence ID No. 3) to accelerate wound healing can readily be appreciated and / or measured with reference to the properties exhibited by injured treated. For present purposes a "treated wound" may be considered to be a wound exposed to a therapeutically effective amount of a medicament of the invention, or which has been treated according to the methods of the invention.

The acceleration of healing of treated wounds may be illustrated by an increased rate of epithelialization compared to control wounds. Thus the methods and medicaments of the invention promote faster reconstitution of a functional epithelial layer over a wounded area that would otherwise be the case.

Alternatively or additionally, accelerated healing of treated wounds can be illustrated by the decreased width compared to control wounds at comparable time points. It will be appreciated that this reduction in wound width ensures that there is a relatively faster rate of wound closure (since there is less width of the wound to be closed) and is indicative of the ability of such drugs to accelerate healing response. Narrow wounds can result in narrower scars that are aesthetically preferable to wider scars.

Accordingly, accelerated wound healing in the context of the present invention should be taken to encompass any increase in healing rate of a treated wound compared to the rate of healing that occurs in treated or untreated control wounds. Preferably the acceleration of wound healing can be assessed against comparing the derithelithelialization rate achieved in treated and control wounds, or comparing the relative width of treated and control wounds at comparable time points. More preferably, accelerated wound healing can be defined as both comprising an increased rate of reepithelialization and a reduction in wound width compared to control wounds at comparable time points.

Preferably the promotion of accelerated wound healing may cause a wound healing rate that is at least 5%, 10%, 20% or 30% higher than the healing rate that occurs in an untreated control wound. More preferably, the promotion of accelerated wound healing may cause a healing rate that is at least 40%, 50% or 60% higher than healing in a control wound. It is even more preferable that promoting accelerated wound healing can cause a healing rate that is at least 70%, 80%, or 90% higher than that occurring in control wounds, and more preferably healing promotion. Accelerated acceleration can cause a healing rate that is at least 100% higher than the rate that occurs in control wounds.

There is a wide range of wound healing disorders that are characterized, or at least partially characterized, by failure, delay, or inappropriate delay in the healing response of the normal wound. The ability of certain methods and medicaments of the invention to promote accelerated wound healing are thus useful in preventing or treating such disorders. Since certain methods and medicaments of the invention are capable of accelerating wound healing through the healing of the wound. In order to promote a stimulated reepithelialization response (thereby increasing the rate at which the wound closes) it is appreciated that these methods and inventions of the invention are particularly advantageous for treating wounds of patients who may otherwise be tend to defective, delayed or otherwise impaired reepithelialization. For example, it is well known that skin wounds in old age exhibit less vigorous reepithelialization response than those of young individuals. There are also many other circumstances or disorders in which wound healing is associated with delayed or otherwise impaired reepithelialization. For example, diabetic patients, polypharmacy patients (eg as a result of advanced age), postmenopausal women, patients susceptible to pressure disorders (eg paraplegics), venous disease patients, clinically obese patients , patients receiving chemotherapy, patients receiving radiotherapy, patients receiving steroid treatment, or immunocompromised patients may all suffer from wound healing with impaired reepithelialization. In many cases the lack of an appropriate reepithelialization response contributes to the development of wound site infections, which may in turn contribute to the formation of chronic wounds such as ulcers. Accordingly, it will be appreciated that such patients are particularly likely to benefit from suitable methods or medicaments of the invention.

Chronic wounds are perhaps the most important example of disorders associated with a delayed wound healing response. A wound can be defined as chronic if it shows no tendency to heal within eight weeks of training when subjected to appropriate (conventional) therapeutic treatment. Well-known examples of chronic wounds include venous ulcers, diabetic ulcers, and decubitus ulcers, but chronic wounds can arise from other normal acute disorders at any time. Typically chronic wounds may arise as a result of wound site infection, inadequate wound treatment, or as a conclusion of progressive tissue disorder caused by metabolic, arterial or venous vascular disease, pressure, radiation damage, outumor.

It will be appreciated that methods and medicaments of the invention capable of accelerating wound healing may be used to treat existing chronic deferrals in order to promote wound healing. Such methods and medications may promote the reepithelialization of chronic wounds, causing desemode, causing healing and closure of the disorder. Preferred methods and medicaments of the invention (such as those using monomeric TGF-β comprising Sequence ID No. 3) may also inhibit wound healing associated wound healing. Scar prevention in such contexts can be particularly advantageous since chronic wounds typically extend over relatively large portions of the patient's body.

In addition, or alternatively, for its use in treating existing chronic deferrals, suitable methods and medicaments of the invention may be used to prevent acute wounds in patients predisposed to healing of weakened wounds developing in chronic wounds.

Since suitable methods and medicaments of the invention are capable of promoting epithelial coverage of the damaged site, they are capable of reducing the likelihood of a treated wound becoming infected. Similarly, this promotion of reepithelialization may benefit the chronic deferred treatment that arises as a result of other conditions such as diabetes or venous disease.

An additional group of patients who may derive particular benefit from the methods and medicaments of the invention are those in which the immune system is compromised (eg, patients undergoing chemotherapy or radiotherapy, or those suffering from HIV infection). It is well recognized that wounds of immunocompromised patients, who may not be able to mount a normal inflammatory response after injury, tend to be associated with poor healing outcomes.

Such patients may benefit from treatment with suitable methods and medications of the invention.

The ability of medicaments and methods of the invention to utilize monomeric TGF-Bs comprising Sequence ID No. 3 to promote wound healing by preventing, reducing or inhibiting scarring is also of use in more general clinical contexts. Examples of these additional benefits may be considered with reference to primary, secondary or tertiary wound healing as described below.

For the purposes of the present invention, primary intention healing may be envisaged to involve closure by surgical means (such as sutures, strips or adhesive clips) of opposite edges of a wound. Primary intention healing is typically employed in the treatment of surgical incisions or other clean wounds, and is associated with minimal levels of tissue loss. The skilled person will recognize that since suitable drugs or methods according to the invention (such as those using monomers comprising Sequence ID N-3) are capable of reducing the width of the wound, they also facilitate the joining of opposite edges of the wound, and thus it may be beneficial in wound healing by primary intention. Further, such methods or medicaments may (as described below) lead to the prevention, reduction or inhibition of scarce that may otherwise occur in such a cure. The inventors believe that treatment thus can have an impact on the macroscopic and microscopic appearance of the scars formed from the treated wounds; macroscopically the scars may be less visible and mingle with the surrounding skin, microscopically the scars may exhibit a regeneration of a more normal skin structure.

For the purposes of the present invention, secondary healing may be considered to constitute wound closure by the wound healing process without direct surgical intervention. Wounds that are cured by secondary intent may be subject to continuous care (eg wound dressing and recurring as well as the application of appropriate medications), but it is the natural processes of granulation tissue deformation and reepithelialization that cause closure. -menting of the wound. It will be appreciated that since the preferred medicaments and methods of the invention (such as those using monomers comprising Sequence ID No. 3) may increase the rate of reepithelialization and diminish subsequent scarring compared to those that occur control wounds. They have utility in promoting wound healing by secondary intention.

Healing by tertiary intent may be considered to comprise surgical closure of a wound that has been previously unopened to allow at least partial formation of the erepithelialization granulation tissue. The properties of the invention's preferred methods and medicaments which make them suitable for use in primary or secondary intention healing are also beneficial in the context of promoting tertiary intention healing.

The use of preferred methods and medicaments of the invention using monomers comprising Sequence ID N-3 to stimulate reepithelialization (as part of their promotion of accelerated wound healing) while inhibiting scarring is also particularly effective in treating associated procedures. graft Treatment using such methods and medicaments of the invention is beneficial to both in a graft donor site (where it may assist in the restoration of a functional epithelial layer by preventing, reducing or inhibiting scar formation), and also at graft target sites (where the effects anti-scar treatment inhibits scar formation, while accelerated healing promotes integration of grafted tissue). The inventors believe that the methods and medicaments of the invention confer advantages in skin contexts using graft, artificial skin, or skin substitutes.

The inventors have found that inventive methods and medicaments utilizing monomers comprising Sequence ID No. 3 can promote accelerated wound healing with scar inhibition while administered prior to wounding, or once a wound has already been formed.

The inventors have found that methods or drugs of the invention utilizing monomeric TGF-B3s, such as those comprising Sequence ID Ne 3, are capable of promoting epithelial regeneration. The promotion of epithelial regeneration within the context of the present invention can be understood to encompass any increase in the rate of epithelial regeneration compared to regeneration that occurs in a treated or untreated control epithelium.

The rate of epithelial regeneration fixed using suitable methods or medicaments according to the invention may readily be compared with that occurring in the treated or untreated control epithelium using any suitable model of epithelial regeneration known in the art. For example, the rate at which sites of experimental epithelial damage having regeneration of known areas can be compared using well-known in vivo models in mice, rats, rabbits or pigs such as those described in Tomlinson and Ferguson (2003), Davidson and others (1991) and Paddock and others (2003).

Without wishing to be bound by any hypothesis, the inventors believe that the promotion of epithelial regeneration achieved by TGF-B3 monomeric forms is mediated by the promotion of epithelial cell migration monomers. Epithelial cells (the migration from which it was promoted) can thus repopulate and regenerate damaged epithelium more rapidly than occurs in the absence of treatment.

It will be appreciated that the promotion of random epithelial regeneration comprising Sequence ID N-3 may be of use to induce effective free-epithelialization in contexts where the reepithelialization response is impaired, inhibited, retarded or otherwise defective. Promotion of epithelial regeneration may also be performed to accelerate the rate of defective or normal epithelial regeneration responses in patients suffering from epithelial damage.

There are many contexts in which the body reepithelialization response may be defective. For example, defective skin reepithelialization is associated with conditions such as pemphigus, Hailey-Hailey disease (benign familial pemphigus), toxic epidermal necrolysis (TEN) / Lyell syndrome, epidermolysis bullosa, cutaneous leishmaniasis, and actinic keratosis. Defective re-pitelialization of the lungs may be associated with idiopathic pulmonary fibrosis (IPF) or interstitial lung disease. Effective rheepithelialization of the eye may be associated with conditions such as partial yimbal stem cell deficiency or corneal erosion. Defective reepithelialization of the colon or gastrointestinal tract may be associated with conditions such as chronic anal fissure (cleft in year), ulcerative colitis or Crohn's disease, and other inflammatory bowel disorders.

As explained above, certain methods or medicaments according to the present invention (and particularly those using monomers comprising Sequence ID No. 3) may prevent, reduce or otherwise inhibit scarring. This scar inhibition can be performed anywhere on the body and in any tissue or organ, including the skin, eye, nerves, tendons, ligaments, muscle, and oral cavity (including lips and the palate) as well as internal organs (such as the liver, the heart, the brain, the abdominal cavity, the pelvic cavity, the thoracic cavity, the intestines, and the reproductive tissue). In the skin, treatment can improve the macroscopic and microscopic appearance of scars; macroscopically scars may be less visible and mingle with the surrounding skin, microscopically collagen fibers within the scar may have the morphology and organization that is most similar to those on the surrounding skin. Scar prevention, reduction or inhibition within the context of the present invention should be understood to address any degree of scar prevention, reduction or inhibition compared to the scar level that occurs in a treated or untreated control wound (such as elsewhere in the descriptive report). Except where the context otherwise requires references to "prevention", "reduction" or "inhibition" of scarring can be taken to be equivalent mechanisms that are all manifested in anti-scar activity.

The prevention, reduction or inhibition of skin scar utilization methods achieved using the methods and medicaments of the invention may be evaluated and / or measured with reference to the microscopic or preferably macroscopic appearance of a treated scar compared to the appearance of an untreated scar. More preferably, scar prevention, reduction or inhibition may be assessed with reference to the macroscopic and microscopic appearance of a treated scar. For present purposes a "treated scar" may be defined as a scar formed in healing a treated wound, whereas an "untreated healing" may be defined as a scar formed in healing an untreated wound, or a wound treated with an untreated wound. placebo or standard care. Suitable comparison scars may preferably be combined with a scar treated with reference to the age, location, size and patient of the scar.

In consideration of the macroscopic appearance of a scar resulting from a treated wound, the extent of the scar, and then the magnitude of any scar prevention, inhibition or reduction achieved, can be assessed with reference to any of several parameters.

Suitable parameters for macroscopic evaluation of screeds may include:

i) Scar color. As noted above, scars can typically be hypopigmented or hyperpigmented with respect to the surrounding skin. Inhibition or scar reduction can be demonstrated when pigmentation of a treated scar is closer to that of unhealed skin than pigmentation of an untreated scar. Similarly, the scars may be redder than the surrounding skin. In this case scar inhibition or reduction may be demonstrated when the redness of a treated scar fades earlier, or more completely, or more closely resembles the appearance of the surrounding skin compared to an untreated scar.

ii) Height of the scar. Scars can typically be high or low compared to the surrounding skin. Inhibition or reduction of scar may be demonstrated when the height of a treated scar is closer to that of unhealed skin (that is, nor is it any higher) than the height of an untreated scar.

iii) Scar surface texture. Scars may have surfaces that are relatively smoother than the surrounding skin (which causes a scar with a "shiny" appearance) or that are rougher than the surrounding skin. Scar inhibition or reduction may be demonstrated when the surface texture of a treated scar is closer to that of unhealed skin than the surface texture of an untreated scar.

iv) Scar rigidity. The abnormal composition and structure of the scars means that it is usually harder than the undamaged skin surrounding the scar. In this case, scar inhibition or reduction may be demonstrated when the stiffness of a treated scar approximates that of the unhealed skin than the stiffness of an untreated scar.

A treated scar will preferably demonstrate prevention, inhibition or scar reduction as assessed by reference to at least one of the parameters for macroscopic assessment set forth above. More preferably a treated scar may demonstrate prevented, inhibited or reduced scar with reference to at least two of the parameters, more preferably to at least three of the parameters, and most preferably all four of these parameters. A total scar assessment can be made using, for example, a Visual Analog Scale or a digital rating scale.

Suitable parameters for microscopic evaluation of scars may include:

(i) Extracellular Matrix (ECM) fiber thickness. Scars typically contain thinner ECM fibers than are found in the surrounding area. This property is even more pronounced in the case of keloid and hypertrophic scars. Inhibition or scar reduction may be demonstrated when the ECM fiber thickness in a treated scar approximates the thickness of ECM fibers found in an unhealed scar than the fiber thickness found in an untreated scar.

ii) ECM fiber orientation. SCF fibers found in scars tend to exhibit a greater degree of alignment with each other than those found in untreated skin (which have a random orientation often referred to as a "basketball hoop"). ECM from pathological scars such as keloids and hypertrophic scars can exhibit even more anomalous orientations, often forming large "swirls" or "capsules" of ECM molecules. Consequently, inhibition or scar reduction may be demonstrated when the orientation of ECM fibers in a treated scar is closer to the orientation of ECM fibers found in unhealed skin than the orientation of such fibers found in an untreated scar.

iii) ECM composition of the scar. The composition of ECM molecules present in scars shows differences from those found in normal scars, with a reduction in the amount of elastin present in ECM scars. Thus scar inhibition or reduction can be demonstrated when the composition of ECM fibers in the dermis of a treated scar approximates the composition of such fibers found in untreated skin than the composition found in an untreated scar. .

iv) Scar cellularity. Scars tend to contain relatively fewer cells than unhealed skin. Accordingly, it will be appreciated that inhibition or scar reduction can be demonstrated when the cellularity of a treated scar is closer to the cellularity of unhealed skin than the cellularity of an untreated scar.

A treated scar will preferably demonstrate scar prevention, reduction or inhibition as assessed with reference to at least one of the parameters for microscopic evaluation set forth above. More preferably a treated scar may demonstrate scar prevention, reduction or inhibition with reference to at least two of the parameters, more preferably at least three of the parameters, and most preferably all four of these parameters.

Prevention, reduction or inhibition of scarring of a treated wound may additionally be evaluated with reference to appropriate parameters used in:

(i) macroscopic clinical evaluation of scars, particularly the evaluation of scars on a subject;

ii) Evaluation of photographic images of scars;

(iii) evaluation of silicone molds or positive plaster casts made of scar silicone molds; and

iv) Microscopic evaluation of scars, for example by histological analysis of the microscopic structure of scars.

It will be appreciated that prevention, reduction or inhibition of healing of a treated wound may be indicated by amelioration of one or more such suitable parameters, and that in the case of prevention, reduction or inhibition as assessed by reference to a number of parameters that these parameters may provide. be combined from different evaluation schemes (eg reduction, inhibition or improvement in at least one parameter used in macroscopic evaluation and at least one parameter used in microscopic evaluation).

Scar prevention, reduction, or inhibition can be demonstrated by an improvement in one or more parameters indicating that a treated scar scarlet approaches the unhealed skin closer to the selected parameters than an untreated scar or scar. control.

Suitable parameters for clinical measurement and evaluation of scars can be selected based on a variety of measurements or evaluations that include those described by Beausang et al. (1998) and Van Zuijlen et al. (2002).

Typically, suitable parameters may include:

1. Evaluation regarding the scoring classification of the Vi-sual Analog Scale (VAS).

Scar prevention, reduction, or inhibition can be demonstrated by a reduction in the VAS score of a treated scar when compared to a control scar. A VAS suitable for use in scar evaluation may be based on the method described by Beausange et al. (1998) .2. Scar Height. scar width, scar perimeter. area of scar scar volume.

The height and width of scars can be measured directly on the subject, for example by using handheld measuring devices such as calipers. Scar width, perimeter and area can be measured on the subject or by image analysis of scar photographs. The skilled person will also be aware of additional noninvasive methods and devices that can be used to investigate suitable parameters, including silicone molding, ultrasound, three-dimensional optical prophylimetry, and high resolution Magnetic Resonance Imaging.

Scar prevention, reduction, or inhibition can be demonstrated by a reduction in the height, width, area, or volume, or any combination thereof, of a treated scar versus an untreated scar.

3. Appearance and / or scar color compared to surrounding unhealed skin.

Appearance or color of a treated scar can be compared to that of the surrounding unhealed skin, and the differences (if any) compared with the difference between the appearance and color of untreated scars and unhealed skin. Such a comparison can be made based on a visual assessment of the respective scars and unhealed skin. The appearance of a scar can be compared to unhealed skin with reference to whether the scar is lighter or darker than unhealed skin. The respective colors of the scars and skin can be perfectly combined with one that is slightly mismatched, obviously mismatched or roughly mismatched.

Alternatively or in addition to visual assessment, there are a number of noninvasive colorimetric devices that can provide data regarding scar pigmentation and unhealed skin, as well as skin redness (which may be an indicator of the degree of vascularity present in the scar or skin). . Examples of such devices include the Chronameter CR-200/300 Minolta; Labscan 600; Dr. Lange Micro Color; Derma specometer; Doppler laser flow meter; and Intracutaneous Spectrophotometric Analysis (SIA) space.

Scar prevention, reduction, or inhibition can be demonstrated by a smaller magnitude difference between the appearance or color of treated scarred and unhealed skin than between untreated and unhealed skin scars.

4. Scar distortion and mechanical performance. Scar distortion can be assessed by visual comparison of a scar and unhealed skin. A proper comparison can classify a selected scar as causing no distortion, mild distortion, moderate distortion, or severe distortion.

The mechanical performance of scars can be evaluated using a number of noninvasive methods and devices based on suction, pressure, torsion, tension and acoustics. Suitable examples included in known devices capable of use in evaluating mechanical performance of scars include Indentometer, Cutometer, Reviscometer, Skin Elastic Analysis, Dermaflex, Durometer, Dermal Torque Meter, Elastometer.

Scar prevention, reduction, or inhibition can be demonstrated by a reduction in the distortion caused by treated scars compared to that caused by untreated scars. It will also be appreciated that scar prevention, reduction or inhibition can be demonstrated by the mechanical performance of untreated skin that is more similar to that treated scars than untreated scars.

5. Scar contour and scar texture. Scar contour can be investigated by visual assessment means. Suitable parameters to consider in such an assessment include whether or not your scar is flush with the surrounding, slightly suppressed, slightly penetrated, hypertrophic or keloid skin. The texture of a scar may be assessed by reference to the appearance of the scar, and it may also be compromised by visual assessment if the scar is, for example, opaque or shiny or has a rough or smooth appearance compared to non-scarred skin. Healed

Scar texture may additionally be assessed with reference to whether the scar has the same texture as unhealed skin (normal texture), is only palpable, firm or hard compared to non-scarred skin. Scar texture can also be assessed with reference to the Hamilton scale (described in Crowe et al. 1998).

In addition to the techniques outlined above, there are a number of noninvasive prophylimetric devices that use optical or mechanical methods to assess scar contour and / or texture. Such evaluations may be performed on the subject's body or, for example, on silicone scar impression impressions, or positive plaster molds made from such impressions.

Scar prevention, reduction, or inhibition can be demonstrated if treated scars have scar profiles and textures more comparable to unhealed skin than untreated scars.

Photographic Evaluations.

Independent Position Panel.

Photographic evaluation of treated and untreated scars can be performed by an independent position panel of advisors who use calibrated and standardized photographs of the scars. Scars may be evaluated by an independent position panel to provide categorical rating data (for example, that a given treated scar is "better", "worse" or "indifferent" when compared to an untreated scar). ) and quantitative data using a Visual Analog Scale (VAS) based on the method described by Beausang et al. (1998). Capturing this data may prepare for the use of appropriate software and / or electronic systems as described in the applicant's co-pending patent application.

Expert Panel

Photographic evaluation of treated and untreated scars may alternatively or additionally be performed by a panel of expert advisors using calibrated and standardized photographs of the scars to be evaluated. The panel of experts may preferably consist of suitable persons skilled in the art such as plastic surgeons and scientists of suitable background.

Such an assessment may provide categorical data as described above or with respect to comparing a time course of selected treated and untreated scarred images.

Appropriate assessments to make may include:

Identification of the best scar, which for the purposes of the present invention may be considered the scar most closely resembling the surrounding skin. Once again the best scar has been identified and the magnitude of the difference between scars can be considered, for example, is the difference between small or obvious scars. Additional parameters that may be considered include the earliest time after scar formation in which a difference in scars can be detected, the time after formation in which the difference in scars is the most obvious (or alternatively the finding that difference continues after the last time point evaluated), as well as considering whether the best scar remained not consistently better.

Consideration may also be given to whether one scar is consistently redder than the other, and whether redness fades over the considered time points (or continues after the last time point) and if so at what time after scar formation. An expert panel can also consider at what time after formation any difference in redness becomes detectable, as well as after time formation when the difference in redness is most obvious.

An expert panel may also consider whether or not one of a treated or untreated cation is consistently whiter than the other, or whiter than unhealed skin. If a whiteness difference is detectable, consideration may be given to the time after scar formation at which the difference can be detected, the time when the difference is most obvious, and the time when the difference disappears.

An additional parameter that can be evaluated by a perforated panel is the texture of treated and untreated scars. In comparing treated and untreated scars the expert panel can consider which scars have the best skin texture, the earliest time after scar formation where any present difference can be detected, the time after formation where any difference is present. most obvious, and the time before any difference disappears.

Comparison of treated and untreated scars may additionally assess which of the scars is the narrowest, and which of the scars is the shortest. Consideration may also be given to the shape of the scar and the proportion of the scar margin that is distinguishable from the surrounding skin. As previously described, visual assessments and color assessments of the presence, degree and location of hyperpigmentation may also be considered.

As noted above, one of the ways in which the quality of treated and untreated scars can be compared is by microscopic evaluation. Microscopic evaluation of scar quality can typically be performed using histological sections of scars. The process of demicroscopically assessing and measuring scars may take into account categorical data based on the following appropriate parameters:

1. Epidermal restitution. Particular attention can be given to the network restoration program, and the thickness of the restored epidermis.

2. Angiogenesis and inflammation. Consideration may be given to the number of blood vessels present, the size of the blood vessels present, and the evidence of inflammation, including an assessment of any inflammation present.

3. Organization of collagen. The reference collagen organization reference can be assessed by the orientation of the collagen fibers present in the scar, the density of such fibers and the thickness of the collagen fiber in the papilla and reticular dermis.

4. Evaluation of the visual analog scale (VAS) of the collagen organization for the papilla dermis and reticular dermis may also provide a useful scar quality index.

5. Other features that may be taken into consideration when evaluating the microscopic quality of scars include scar elevation or depression relative to surrounding unhealed skin, and the prominence or visibility of the scar at the normal skin interface.

6. It will be appreciated that the assessments described above allow the generation of scar classification data that may not provide an indication of whether a treated scar is better, worse or indifferent compared to a control, untreated scar or other scar for proper comparison.

In addition to categorical data, quantitative data (preferably over the above parameters) can be generated using image analysis in combination with appropriate visualization techniques. Examples of suitable visualization techniques that can be employed to assess scar quality are specific histological or immunotagging stains, where the degree of staining or labeling present can be quantitatively determined by image analysis.

Quantitative data can be produced usefully and readily with respect to the following parameters:

1. Width, height, elevation, volume and scar area.

2. Epithelial thickness and coverage (eg, the area of the epidermis present in a scar or the proportion of a wound with epidermal coverage).

3. Number, size, area (ie cross section) and location of blood vessels.

4. Degree of inflammation, number, location and populations / types of inflammatory cells present.

5. Collagen organization, collagen fiber thickness, collagen fiber density.

Scar prevention, reduction, or inhibition can be demonstrated by a change in any of the parameters considered above such that a treated scar resembles unhealed skin more than a control or untreated scar (or other appropriate comparator).

The evaluations and parameters discussed are suitable for comparisons of the effects of peptides or medicaments of the invention compared to control, placebo or standard care treatment in animals or humans. Appropriate statistical tests can be used to analyze data sets generated from different treatments in order to investigate the meaning of the results.

Preferably scar prevention, reduction or inhibition may be demonstrated by reference to more than one parameter. More preferably, prevention, reduction or inhibition of scarring may be demonstrated with reference (i.e. observed in the subject) to a clinical parameter and a photographic parameter. Even more preferably scar prevention, reduction or inhibition can be demonstrated by reference to a clinical parameter, a photographic parameter, and also to a microscopic evaluation parameter (e.g., a histological parameter). More preferably, scar prevention, reduction or inhibition can be demonstrated with reference to a clinical classification of VAS1 index and external position panel VAS classification (from photographic images) and reticular dermal VASmicroscopic index.

The use of suitable methods and medicament of the invention is capable of causing a rapid improvement in the cosmetic appearance of a then treated wound area. Cosmetic considerations are important in various clinical contexts, particularly when wounds are formed on prominent body sites such as the face, neck, and hands. Accordingly, scar inhibition (which may preferably be in combination with accelerated wound healing) at such locations where it is desired to improve the cosmetic appearance of the scar formed represents a preferred embodiment of the invention.

In addition to its cosmetic impact, skin scarring is responsible for a number of deleterious effects that afflict those suffering from talc healing. For example, skin scarring may be associated with reduced physical and mechanical function, particularly in the case of contractile scarring (such as hypertrophic scarring) and / or situations where scarring is formed through the joints. In these cases the altered mechanical properties of scarred skin, as opposed to unhealed skin, and the effects of scar contraction can lead to dramatically restricted movement of a joint as soon as it is performed. Accordingly, it is a preferred embodiment that suitable drugs and methods of the invention be used to prevent, reduce or inhibit scarring of body-covering wounds (preferably also accelerating the healing of such wounds). In another preferred embodiment suitable medicaments and methods of the invention may be used to promote accelerated wound healing and / or prevent, reduce or inhibit wound healing at greater risk of deforming a contractile scar.

The extent of scar formation, and thus the extent of cosmetic damage or other damage that may be caused by the scar, may also be influenced by factors such as the tension of the site at which it is formed. For example, it is known that skin under relatively high tension (such as that extending over the chest, or associated with tension lines) may be inclined to form darker scars than elsewhere on the body. Thus in a preferred embodiment suitable medicaments and methods of the invention may be used to promote accelerated wound healing and / or to prevent, reduce or inhibit wound healing at sites of high skin tension. There are many surgical procedures that can be used in scar revision to allow wound and scar realignment such that they are subject to reduced tension. Probably the best known of these is "Z-plasty" in two V-shaped skin flaps that are transposed to allow rotation of a tension line. Thus in a more preferred embodiment such medicaments and methods of the invention are used to promote accelerated healing and / or to prevent, reduce or inhibit wound healing during the surgical review of disfigured scars.

Pathological healing may have more pronounced detrimental effects than those that arise even as a result of relatively severe normal scarring. Common examples of pathological scars include keloids and hypertrophic scars. It is recognized that certain wound types, or certain individuals may be predisposed to pathological scarring. For example, individuals of Afro-Caribbean, Japanese or Mongoloid heritage, or those who have a family history of pathological scarring may be considered to be at greater risk of keloid or hypertrophic scar formation. Wounds in children, particularly burn wounds in children, are also associated with increased hypertrophic scar formation. Accordingly, it is a preferred embodiment of the invention that suitable drugs and methods are used to promote accelerated wound healing and / or prevent, reduce or inhibit wound healing in which there is an increased risk of pathological scar formation.

Although individuals already subject to pathological scarring suffer from a predisposition to additional excessive scarring, there is often a clinical need to surgically review keloids or hypertrophic scars, with a present risk of consequent pathological scarring. Thus it is a further preferred embodiment of the invention that suitable drugs and methods be used to promote accelerated wound healing and / or prevent, reduce or inhibit wound healing produced by surgical review of pathological scars.

It is recognized that wounds resulting from hard burn injuries (which for the purposes of the present invention may also be taken to cover burn injuries involving hot liquids or gases) may extend over large areas of a then afflicted individual. As a result, burns can cause scar formation by covering a large proportion of a patient's body, thereby increasing the risk that the scar formed will cover areas of high cosmetic importance (such as the face, neck, arms or ears). hands) or of mechanical importance (particularly the covering or surrounding regions of the joints). Hot liquid burn injuries are often sustained by children (eg as a result of spilled frying pans, kettles or the like) and, due to the relatively smaller body size of children, are particularly likely to cause extensive damage to a large proportion of the body area. . It is a further preferred embodiment of the invention that suitable drugs and methods are used to promote accelerated wound healing and / or to prevent, reduce or inhibit wound healing produced by burn injuries.

As noted above, wound healing in response to burn injuries is often associated with adverse healing outcomes such as the formation of hypertrophic scars. An additional consequence of the relatively large size of burn injuries is that they are particularly susceptible to complications such as infection and desiccation arising from the lack of a functional epithelial layer.

In view of the foregoing it will be appreciated that the appropriate methods and medicaments of the invention may be used in the treatment of burn wounds to reduce the level of scarring that occurs as a result of wound and / or acceleration of wound reconstruction. an epithelial-functional barrier.

The inventors have found that inventive methods and medicaments utilizing monomers such as those comprising Sequence ID Ns 3 are capable of promoting reepithelialization. Consequently such methods and medications are particularly effective in treating all wounds involving damage to the epithelial layer. Such injuries are exemplified, but not limited to, skin wounds where the epidermis is damaged. It will be appreciated, however, that such methods and inventions of the invention are also applicable to other types of wounds without damage to the epithelium, such as injuries involving the epitheliorespiratory, digestive epithelium, or epithelium involving internal tissues or organs (such as the epithelium of the epithelium). peritoneum).

Healing wounds involving the peritoneum (the epithelial covering of the internal organs and / or the interior of the body cavity) can often cause adhesions. Such adhesions are a common conclusion of surgery involving gynecological or intestinal tissues. The inventors believe that the ability of the methods and medicaments of the invention (using suitable monomers such as those comprising Sequence ID N93) to accelerate peritoneum regeneration by reducing scarring may reduce the incidence of improper attachment of portions of one peritoneum to another, and thereby thus reduce the occurrence of adhesions. Accordingly, the use of the methods and medicaments of the invention to prevent the formation of intestinal or gynecological adhesions represents a preferred embodiment of the invention. Certainly the use of such methods or medicaments of the invention in healing all wounds involving the peritoneum is a preferred embodiment.

The methods or medicaments of the invention may be used prophylactically, for example, where no wound exists but where a wound that would otherwise give rise to a chronic wound scar must be formed. As an example, drugs according to the invention may be administered to sites that are under injury as a result of elective procedures (such as surgery), or to locations believed to be at high risk of injury. It may be preferred that the medicaments of the invention be administered to the site approximately at the time of the wound, or just prior to the formation of a wound (e.g., up to six hours prior to wounding), or the medicaments may be administered in a single dose. hour prior to the injury (eg, up to 48 hours before a wound is formed). The skilled person will appreciate that the most preferred times of administration prior to the formation of a wound will be determined by reference to a number of factors including formulation and route of administration of the selected medication, the dosage of the drug to be administered, the size and nature of the wound to be formed, and the biological status of the patient (which may be determined with reference to factors such as age, health , and patient predisposition to healing or scarring complications ad versa). Prophylactic use of methods and medicaments according to the invention is a preferred embodiment of the invention, and is particularly preferred in promoting accelerated wound healing and / or prevention, reduction or inhibition of scarring in the context of surgical wounds.

The methods and medicaments of the invention are also capable of promoting accelerated healing and / or inhibited wound healing if administered after a wound has been formed. It is preferred that such administration should occur as soon as possible after wound formation, but the agents of the invention are capable of promoting accelerated wound healing and / or preventing, reducing or inhibiting scarring at any time above until the healing process is completed ( that is even in case of an already partially healing wound the methods and medicaments of the invention may be used to promote accelerated wound healing and / or to prevent, reduce or inhibit scarring with respect to the remaining uncured portion). It will be appreciated that the "window" in which the methods and medicaments of the invention may be used to promote accelerated healing and / or prevent, reduce or inhibit wound healing is dependent upon the nature of the wound in question (which includes the degree of wound healing). damage occurred, and the size of the wounded area). Thus in the case of a large wound the methods and medicaments of the invention may be administered relatively late in the healing response while still promoting accelerated healing and / or preventing, reducing or inhibiting wound healing. The methods and medicaments of the invention may, for example, preferably be administered within the first 24 hours after a wound is formed, but may further promote accelerated wound healing and / or prevent, reduce or inhibit scarring if administered up to ten or more. days after the injury.

The methods and medicaments of the invention may be administered on one or more occasions as necessary to promote accelerated wound healing and / or to prevent, reduce or inhibit scarring. For example, therapeutically effective amounts of the medicaments may be administered to a wound as often as necessary until the healing process is over. As an example, the inventive drugs may be administered daily or twice daily at a rate for at least the first three days following wound formation. The inventors have found that diets involving two administrations of medicaments of the invention, the first before the formation of a wound and the second after the wound, are particularly beneficial in reducing scar deformation. Preferably such diets may involve a first administration immediately prior to wound formation and a second administration 24 hours after injury.

More preferably, the methods or medicaments of the invention may both be administered before and after wound formation. The inventors have found that administering medicaments of the invention immediately prior to wound formation, followed by daily administration of such agents. In the days following the injury, it is particularly effective in promoting accelerated wound healing and / or preventing, reducing inhibiting scarring.

For purposes of this disclosure "agent" or "agent of the invention" means biologically or therapeutically active monomeric TGF-β. Such agents may preferably be jungle-type TGF-Bs, and more preferably may be monomeric TGF-Bs capable of promoting accelerated wound healing and / or inhibiting scarring, of which a preferred example comprises the monomers prepared in Sequence ID No. 3. It will be appreciated that all such agents may be incorporated into medicaments according to the invention, and may be used in the methods or methods of the invention.

It will be appreciated that the amount of a medicament of the invention to be applied to a wound depends on a number of factors such as biological activity and the bioavailability of the agent present in the medicament, which in turn depends on, among other factors, the nature of the agent and the mode of administration of the medicament. Other factors in determining an appropriate therapeutic amount of a drug may include:

A) The half-life of the agent in the subject being treated.

Β) The specific condition to be treated (eg acute wound or chronic wounds).

C) The age of the subject. Frequency of administration will also be influenced by the above mentioned factors and particularly by the half-life of the agent chosen within the subject being treated.

Generally, when medicaments according to the invention are used to treat existing wounds, the medicament should be administered as soon as the wound occurs (or in the case of wounds that are not immediately apparent as those internally on the body as soon as the wound is measured). diagnosed). Therapy with methods or medicaments according to the invention should continue until the healing process is accelerated, and / or scar prevented, reduced or inhibited, to the satisfaction of the clinician.

The frequency of administration will depend on the biological agent half-life used. Typically a cream or ointment containing a person of the invention should be administered to a target tissue such that the concentration of the agent in a wound is maintained at an appropriate level for therapeutic effect. This may require daily administration or even several times a day.

Medicaments of the invention may be administered by any suitable route capable of achieving the desired effect of promoting wound healing and / or preventing, reducing or inhibiting scarring, but it is preferred that the medicaments be administered locally to a wound site.

The inventors have found that the promotion of accelerated wound healing and / or scar prevention, reduction or inhibition can be accomplished by administering an agent of the invention by injection into the wound site.

For example, in the case of skin wounds, the agents of the invention may be administered by intradermal injection. Thus a preferred medicament according to the invention comprises an injectable solution of an agent of the invention (for example, for injection around the edges of an epithelial damage site or a site which is likely to be damaged). Formulations suitable for use in this embodiment of the invention are considered below.

Alternatively or additionally, the medicaments of the invention may also be administered in a topical form to promote accelerated wound healing and / or to prevent, reduce or inhibit scarring. Such administration may be effected as part of the initial and / or continuing care for the injured area.

The inventors have found that the promotion of accelerated wound healing and / or prevention, reduction or inhibition of scarring is particularly improved by topically applying an agent of the invention to a wound (or, in the case of prophylactic application, to a tissue or site where a wound is present). be formed).

Compositions or medicaments containing agents of the invention may take a number of different forms depending upon the particular manner in which they are to be used. Thus, for example, they may be in the form of a liquid, ointment, cream, gel, hydrogel, powder or aerosol. Other compositions are suitable for topical treatment of a wound as a preferred means of administering agents of the invention to a subject (person or animal) in need of treatment.

The agents of the invention may be provided in a sterile bandage or bandage which may be used to cover a wound or other site of epithelial damage to be treated.

It will be appreciated that the carrier of a composition comprising agents of the invention should be one which is well tolerated by the patient and allows release of the agent to the wound. Such carrier is preferably biodegradable, biosoluble, bioresorbable and / or non-inflammatory.

Medicines and compositions comprising inventive agents may be used in various ways. Thus, for example, a composition may be applied to and / or around a wound in order to promote accelerated wound healing and / or to prevent, reduce or inhibit scarring. If the patch is to be applied to an "existing" wound, then the pharmaceutically acceptable carrier will be one that is relatively "soft" ie a vehicle that is biocompatible, biodegradable, biosoluble and non-inflammatory.

An agent of the invention, or a talagent-encoding nucleic acid (as considered below), may be incorporated into a slow or prolonged release device. Such devices may, for example, be placed on or inserted under the skin and the nucleic acid or agent may be released for days, weeks or even months. Such a device may be particularly useful for patients (such as those suffering from chronic wounds) who require long-term promotion of accelerated wound healing and / or scar prevention, reduction or inhibition. Devices may be particularly advantageous when used for the administration of an agent or a nucleic acid that would normally require frequent administration (e.g., at least daily administration by other routes).

Daily doses of an agent of the invention may be given as a single administration (e.g., a daily application of a topical formulation or a daily injection). Alternatively, the inventive agent may require administration two or more times during a day. As an additional alternative, a slow release device may be used to provide optimal doses of an agent of the invention to a patient without the need to administer repeated doses. .

In one embodiment a pharmaceutical carrier for administration of an agent of the invention may be a liquid and a pharmaceutically suitable composition would be in the form of a solution. In another embodiment, the pharmaceutically acceptable carrier is a solid and a suitable composition is in the form of a powder or tablets. In a further embodiment the agent of the invention may be formulated as a pharmaceutically acceptable transdermal patch part.

A solid carrier may include one or more substances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, sliding agents, pressure banding, binders or tablet disintegrating agents; it may also be an encapsulation material. In powders, the carrier is a finely divided solid which is mixed with the finely divided agent of the invention. In tablets, the agent of the invention is mixed with a vehicle having the necessary compression properties in appropriate proportions, and compressed into the desired shape and size. The powders and tablets preferably contain up to 99% of the agent of the invention. Suitable carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melt waxes and ion exchange resins.

Liquid carriers may be used to prepare solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The agent of the invention may be dissolved or suspended in a pharmaceutically acceptable carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid carrier may contain other pharmaceutically suitable additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity adjusters, stabilizers or osmoregulators. Suitable examples of liquid carriers for oral and parenteral administration include water (which partially contain additives as above, for example cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, for example pio, glycols) and their derivatives, and oils (eg fractionated coconut oil and peanut oil). It may be generally preferred for medicaments or methods of the invention to use formulations wherein monomeric TGF-Bs, such as TGF-B3, are provided in the absence of alcohols. Such alcohol-free formulations may particularly be preferred for use in deferred sites (or places where wounds should be formed) due to their relatively "mild" nature. For parenteral administration, the carrier may also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid carrier for pressurized compositions may be halogenated hydrocarbon or other pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions which are sterile solutions or suspensions may be used by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intradermal, intrastromal (corneal) or asubcutaneous injection. Sterile solutions may also be administered intravenously. The agent of the invention may be prepared as a sterile solid composition which may be dissolved or suspended at the time of administration using sterile water, saline, or other suitable sterile injectable medium. It is intended to include inert and necessary binder vehicles, suspending agents, lubricants and preservatives

In the situation where it is desired to administer an agent of the invention by oral ingestion, it will be appreciated that the agent chosen will preferably be an agent having a high degree of resistance to degradation. For example, the agent of the invention may be protected (for example, using the techniques described above) so that its rate of digestive tract degradation is reduced.

Compositions of agents of the invention are suitable for use to promote accelerated wound healing and / or inhibit corneal scarring. Corneal wounds may result from trauma to the eye arising as a result of accidental injury (as considered above) or as a result of surgical operations (eg, corneal laser surgery). In this case a preferred medicament of the invention may be in the form of an eye drop.

Agents of the invention may be used on a scale of "internal" wounds (ie wounds that occur inside the body rather than on an external surface). Thus, for example, the drugs according to the invention may be formulated for inhalation for use in wounds arising in the lungs or other respiratory epithelium.

Known procedures such as those conventionally employed by the pharmaceutical industry (e.g., inventive experiments, clinical trials, etc.) may be used to establish specific formulations of compositions comprising agents of the invention and precise therapeutic paradigms for administering such compositions (such as daily doses). of the active agent and the frequency of administration.) An appropriate daily dose of an inventive agent capable of accelerating wound healing and / or preventing, reducing or inhibiting scarring depends on a range of factors including (but unlimited (a) the nature of the wound tissue, area and / or depth of the wound to be treated, the severity of the wound, and the presence or absence of factors predisposed to pathological scarring or chronic wound formation.

As an example, the amount of an active agent that can be administered to a wound or site of epithelial damage in a single treatment incidence may preferably be in the region of 50-200ng / cm linear of the wound or cm2 of epithelial damage (if administered by injection), or 100-300ng / cm linear wound or cm2 epithelial damage (if administered topically).

For a further example, the preferred amount of an active agent to be administered daily to a wound or epithelial damage site may be in the region of 50ng / cm linear wound or cm2 epithelial damage (administered by injection), or 100ng / cm2. cm linear wound or cm2 epithelial damage (if administered topically).

As an example, the total amount of an active agent that can be administered by local injection to a wound or site of epithelial damage may preferably be in the region of 50ng / 100ML per linear centimeter of wound or cm2 of epithelial damage, given a once a day for up to three days, desemodo, providing a total dose of 150ng / cm linear wound or cm2 epithelial damage.

In the case of topical treatment of wounds or acute epithelial damage sites, an appropriate amount of an active agent may preferably be in the region of 100ng / linear wound or cm2 or epithelial damage given once daily for up to 3 days, thereby providing a total dose of 300ng / cm linear wound or cm2 epithelial damage.

Without decreasing the above, the amount of an agent according to the invention required for the treatment of wounds or other epithelial damage sites will typically be within the range of 1 pg to 1 mg of the agent administered per linear centimeter of wound or epithelial damage per 24 hours. although this figure can be modified up or down in response to the factors outlined above. The agent may preferably be provided as a solution of lpg / ΙΟΟμΙ -1 mg / 1 OOpL of the agent, and 100μL of such solution administered per linear centimeter of wound or epithelial damage over a period of 24 hours.

The agent may more preferably be administered as a 10pg / 100pL - 100pg / 100pL solution with 100μΙ of such solution administered per linear centimeter of wound or epithelial damage over a 24 hour period.

More preferably the agent may be administered as a solution of 1ng / 100pL - 100ng / 10OML with 100µl of such solution administered per linear centimeter of wound or epithelial damage over a 24 hour period.

Generally, compositions comprising agents of the invention should be formulated such that when administered to a wound an agent concentration of between 0.79 mM and 0.79 mM per deferred linear centimeter or epithelial damage is achieved. Preferably the agent may be delivered at concentrations between 7.9pM and 0.079mM per linear centimeter.

An agent of the invention (such as Sequence ID N-3 peptide) may be administered at a concentration between 0.79pM and 0.79mM. Preferably an agent of the invention may be administered at a concentration between 7.9pM and 0.079 mM More preferably an inventive agent may be administered at a concentration between 0.79nM and 0.79μΜ.

Purely as an example, an injectable solution containing between 10pg / 100μL- and 100μg / 100μL of an agent of the invention (such as Sequence ID Ng 3 peptide) is suitable for the application to promote accelerated dermal wound healing and / or scar inhibition. when administered as an intradermal injection and dosed with 100μΙ_ per linear cm of wound margin.

In the case of a monomeric peptide of Sequence ID No. 53, preferred dosages for administration to a wound may be in the region of 1 ng / 100 µL - 1000ng / 100 µL, and 100 µL of such a solution administered per cm 2/2 wound margin. .

The agents of the invention may be used to promote accelerated wound healing and / or prevent, reduce or inhibit scarring as a monotherapy (for example, by using the medicaments of the invention alone). Alternatively, the methods or medicaments of the invention may be used in combination with other compounds or treatments for promoting wound healing or scar inhibition. Suitable treatments that may be used as part of such combination therapies will be known to those skilled in the art.

The inventors have found that medicaments according to the present invention, and particularly those comprising Sequence ID N-3 peptides may advantageously be formulated in the presence of a sugar. This sugar may be a reducing or non-reducing sugar and / or a phosphate or phosphonate derived therefrom. Examples of such sugars may be selected from, but are not limited to, the group comprising maltose, mannose, trehalose, arabinose, mannitol, sucrose, fructose, dextrose and glucose. Preferred sugars may be selected from the group consisting of maltose and trehalose. The medicaments of the invention, and particularly those according to this embodiment of the invention, may preferably have a pH between 5 and 7.

It will be appreciated that monomeric TGF-β peptides may represent favorable agents to be administered by techniques involving cellular expression of nucleic acid sequences encoding such molecules. Such methods of cellular expression are particularly suitable for medical use where the therapeutic effects of peptides are required over an extended period, for example, in contexts where it is desirable to increase an otherwise defective wound healing response over a period of time. It is particularly preferred that TGF-β monomers to be administered via cellular expression comprise peptides defined by Sequence ID Ns 3.

Many known methods of delivering peptide agents of the invention to tissues such as wounds have the disadvantage that sustained levels of agent of the invention may be difficult to achieve at the site of treatment over the course of even a few days because peptide agents may be half-full. -small life in vivo. Agent half-lives can be short for several reasons including:

(i) Protease degradation and the like.

(ii) Removal of binding proteins.

(iii) Binding and inhibition of agent activity by extracellular matrix molecules.

In addition, agents used to promote accelerated healing and / or scar prevention, reduction or inhibition need to be administered in a suitable vehicle and are often provided as a composition comprising the agent and the vehicle. As discussed, such vehicles are preferably non-inflammatory, biocompatible, bioresorbable and should not degrade or inactivate the agent (on storage or otherwise). However, it can often be difficult to provide a satisfactory vehicle to deliver agents to tissue with a wound to be treated.

A convenient way in which these problems can be prevented or mitigated is to provide a therapeutically effective amount of an agent of the invention in an area to be treated by degene therapy.

Due to the degeneration of the genetic code, it is clear that the nucleic acid sequences encoding the agents suitable for use according to the invention may be varied or changed without substantially affecting the sequence of the encoded product, thereby providing a variation. of the same. Possible nucleic acid sequences that can be used to encode peptides defined by Sequence ID Ne 1 to 3 will be readily apparent to the skilled person, and suitable examples are provided as Sequence ID Ns 4 to 7 respectively.

Gene therapy delivery systems are highly suitable for achieving sustained levels of an agent of the invention over a longer period of time than is possible for most conventional delivery systems. Agents of the invention suitable for promoting accelerated wound healing and / or inhibited scarring may be continuously expressed from cells at a wound site that has been transformed with the DNA molecule described in the ninth aspect of the invention. Accordingly, even if the agent of the invention had a very short half-life in vivo, therapeutically effective amounts of the person can be continuously expressed from the treated tissue.

In addition, gene therapy delivery systems may be used to deliver the DNA molecule (and thus the inventive agent) without the need to use conventional pharmaceutical carriers such as those required for ointments or creams that come in contact with. the wound.

A suitable gene therapy delivery system is preferably such that the DNA molecule is capable of being expressed (when the delivery system is administered to a patient) to produce a peptide defined by the group comprising Sequence ID No. 1 to 1. 3. The DNA molecule may be contained within a suitable vector to form a recombinant vector. The vector may, for example, be a plasmid, cosmid or phage. Such recombinant vectors are highly useful in gene therapy delivery systems of the invention for transformation cells with suitable DNA molecules.

Recombinant vectors may also include other functional elements. For example, recombinant vectors may be designed such that the vector will replicate autonomously in the cell nucleus. In this case, elements that induce DNA replication may be required in the recombining vector. Alternatively the recombinant vector may be designed such that the vector and recombinant DNA molecule integrate into the genome of a cell. In this case DNA sequences that favor target integration (e.g., by homologous recombination) are desirable. Recombinant vectors may also have DNA encoding for genes that may be used as selectable markers in the cloning process.

The recombinant vector may also further comprise a promoter or regulator for controlling expression of the required gene.

The DNA molecule may (but not necessarily) be one that becomes incorporated into the DNA of cells of the subject being treated. Undifferentiated cells can be stably transformed leading to the production of genetically modified daughter cells. When this is the case, expression regulation in the subject may be required, for example, with specific transcription factors, gene activators or more preferably with inducible promoters that transcribe the gene in response to a signal specifically found in a wound. Alternatively, the delivery system may be designed to favor unstable or transient transformation of differentiated cells in the subject being treated. In this example, expression regulation may be less important because expression of the DNA molecule will stop when transformed cells die or stop expressing the protein (ideally when promoting accelerated healing of the reduced scar wound is effected). ).

The delivery system can provide the DNA molecule to a subject without incorporating it into a vector. For example, the DNA molecule may be incorporated into a liposome or virus particle.

Alternatively, the "naked" DNA molecule may be introduced into a subject's cell by suitable means, for example, direct endocytotic absorption.

The DNA molecule may be transferred to the cells of a subject to be treated by transfection, infection, microinjection, cell fusion, protoplast fusion or ballistic bombardment. For example, transfer may be by ballistic transfection with coated gold particles, liposomes containing the DNA molecule, viral vectors (e.g., adenovirus), and means of providing direct DNA uptake (e.g., endocytosis) by applying plasmid DNA directly. to a topical or injection wound.

Cell expression of the agent of the invention may be by cells at the edge of the undamaged area surrounding the wound, or may alternatively be by cells introduced therapeutically into the wound (e.g., endogenous or exogenous cultured cells involved in the wound healing response). ).

It will be appreciated that cells that must be therapeutically introduced to promote accelerated wound healing and / or prevention, reduction or inhibition of scarring can be engineered ex vivo such that they express increased levels of an agent of the invention, and then introduced into the wounded area. Such cells may preferably be cells cultured ex vivo for use in the preparation or manufacture of artificial or replacement skin for use in promoting wound healing. The cells may most preferably be autologous cells, although it will be appreciated that all suitable cells may be used.

Accordingly, in a seventeenth aspect of the invention, there is provided a medicament comprising cells induced to express an agent of the present invention. Such cells may preferably express a monomeric TGF-B3.

Induction of cellular expression of an agent of the invention may be effected by means of incorporation into nucleic acid cells causing expression of agents suitable for use according to the invention.

The invention will now be further described by way of example with reference to the following protocols and experimental studies, and to the figures in which:

Figure 1 illustrates the comparison results of monomeric and dimeric forms of TGF-B3 by SDS-PAGE under reduced and unreduced conditions;

Figure 2 shows the model used to position incisional wounds in experimental rats;

Figure 3 summarizes treatments administered at experimental animal wound sites;

Figure 4 shows the results of the evaluation, three days after offering, of the appearance of experimental wounds treated with dimeric or monomeric TGF-B3 Figure 5 summarizes treatments administered at experimental animal wound sites;

Figure 6 shows the results of the evaluation, 70 days after injury, of the appearance of experimental scars treated with monomeric or dimeric TGF-B3;

Figure 7 shows representative macroscopic images showing the appearance of experimental scars 70 days after injury;

Figure 8 Representative microscopic images showing the structure of experimental scars 70 days after injury;

Figure 9 illustrates the production of TGF-B3 monomers by successfully transformed host cells;

Figure 10 illustrates the generation of dimeric and monomeric TGF-B3 under experimental protein refolding conditions; and

Figure 11 illustrates the isolation of TGF-B3 proteins by hydrophobic interaction / ultrafiltration chromatography.

Information regarding the amino acid or nucleic acid sequences of importance is provided in the "Sequence Information" section (including amino acid sequences of the active human TGF-β isoform fragments suitable for use according to the invention, as well as the molecule sequences. DNA encoding complete peptides from the TGF-β isoforms, and DNA encoding the active wild type TGF-B3 fragment, and the primer DNA sequences used in cDNA generation encoding human TGF-β isoforms suitable for use in accordance with the invention).

PROTOCOLS AND EXAMPLES.

1. METHODOLOGIES FOR PRODUCTION. REDOBRAMENT, AND PURIFYING OF MONOMERIC TGF-β PROTEINS.

1.1 Nucleotide Sequences

DNA sequences encoding complete TGF-β proteins are shown as Sequence ID N9 4 to 6. Complete TGF-β proteins consist of a signal peptide (shown in italics), propeptide (shown in bold) as well as the active fragment ( shown in textonormal). Nucleotide sequence coding for active wild type TGF-33 fragments is shown in Sequence ID No. 7.

1.2 cDNA Generation

Total RNA from an incisional human wound (taken 5 days after the wound) was treated with "free DNA" (Ambion) to remove any DNA contamination. Using total RNA as a model, cDNA for each of the three mammalian TGF-β isoforms, namely TGF-B1, TGF-32 and TGF-B3 was generated by reverse polymerase-transcriptase chain reaction (RT-PCR). ). The RT-PCR master mix was prepared from the Brilliant® QRT-PCR Core Reagent Kit, 1 step (Stra-tagene). One microgram of RNA was added to 50 μΙ_ of a solution containing: One-step QRT-PCR buffer, 0.2mM dNTPs, 3.5mM MgCl2, 1pL Stratascript reverse transcriptase, 2.5 units TaqPo-limerase, 0, 4μΙ_ Sense Primer (Figure 1), 0.4μΜ antisense initiator, (as shown in Sequence Information). The reaction was placed in a thermal cycler (Hybaid PCR Expresses) and performed under the following conditions: 30 min at 45 ° C, 10 min at 95 ° C, then 40 cycles of 95 ° C for 30s, 65 ° C for 1 min and 72 ° C for 1 min. The final step is 72 ° C for 10 min. PCR samples were performed on 2% (w / v) agarose gels to verify lane size and purified using Wizard PCR Prep Kit (Promega).

1.3 Plasmid Construction

The pET-3d vector used is derived from the pBR322 vector and contains a T7 engine under LacUV 5 control and an am picillin resistant marker gene.

The three TGF-β isoform DNA fragments (generated in section 1.2) were subcloned into pET-3d vectors at the H1 Bam and Nco I sites (5'-3 'respectively). The resulting ligations were then transformed into XL10 Gold cells (Stratagene) and PCR colony analysis was performed to locate clones containing an insert. Final clones were grown from their extracted plasmid DNA in water using Qiaprep®Spin Mini-prep Kit (Qiagen). Plasmids were sequenced and verified using T7 promoter primer (5'-TAA GAC TCA CTA TAG GG-3 ') and T7 terminal primer (5'-GTC AGT TAT TGC TCA GCG G-3').

1.4 Transformation and Cloning

10 μL (50 ng per μΙ) of the plasmid DNA coding for one of the TGF-β isoforms (from section 1.3) was added to 1mL of cold competent E.coli BL21 (DE3) Singles ™ pLysS (Novagen) cells (4 ° Ç). After 20 min the cells were heated and hatched by incubation for 30s at 42 ° C in a water bath. 100µl of Psi medium was added to the cell / plasmid mixture and stirred at 37 ° C for 90 min. Aliquots of 50μL and 100μL were plated on LB agar plates containing 100μg / mL ampicillin (Sigma) and incubated for 18 hours at 37 ° C. Single colonies were cultured and frozen cell stocks generated and stored at -80 ° C. Plasmid DNA was analyzed from cell stocks to verify correct transformation.

1.5 Expression

Frozen E. coli cell ampoules that were transformed with one of the TGF-β isoforms (from section 1.4) were recovered and inoculated into deflector Erlenmeyer flasks containing 100mL of Ampicillin LBe media and 100μg / mL. The flasks were incubated with shaking overnight at 37 ° C. 5mL of this overnight culture was added to a 2 liter Erlenmeyer flask (500mL LB / amp media) and incubated with shaking at 37 ° C. 2mL of broth samples were taken hourly to track growth and TGF-μ isoform (post-induction) expression. Growth was determined by measuring absorbance on a spectrophotometer at a wavelength of 600nm. When absorbance measured 0.6 Abs the cells were induced to express TGF-β isoforms by the addition of isopropyl β-D-Thiogalctopyroside (IPTG, Sigma) to a final concentration of 1 mM. Cultures were incubated for an additional 4 hours. The 0.5mL of broth samples were pelleted by centrifugation (10min 10,000 rpm in a Sorvall Biofuge) and the supernatant discharged. The pellet was resuspended in 50µl of sodium dodecyl sulfate (SDS) - polyacrylamide gel-electrophoresis (PAGE) sample buffer and heated for 10 minutes in a 95 ° C water bath. 10μΙ samples were loaded on SDS-PAGE. SDS-PAGE and Co-omassie Blue staining were performed (as described in A.T Andrews, 1986) using a HoefertDMighty Small SE 245 Dual Gel Caster (Amersham). The gels were 1mm thick and contained 15% (v / v) polyacrylamide gel.

1.6 Cell Harvesting and Insulation of Inclusion Bodies

Section 1.5 cells were pelleted by centrifuging 5000g for 10 min at 4 ° C in a Hettich Routine 46R centrifuge with a Rotor 4315. Cell disruption and recovery of each (inclusion bodies) from insoluble TGF-β isoforms were performed at 4 ° C. ° C. Cells were suspended in 50mL 100mM Tris / HCI (Sigma), 10mM EDTA (Sigma) pH 8.3 and were stopped by sonication using a Sanjo So-niprep 150. 0.2% (w / w) Triton X -100 (Sigma) was added to the suspension and stirred for one hour. The suspension was centrifuged at 12,000g for 40 min. The pellets were resuspended in 50mL 100mMTris / HCl, 10mM EDTApH 8.3 at room temperature before being centrifuged for 40 min in 15,000g.

1.7 Solubilization of Inclusion Bodies

The section 1.6 pellets were resuspended in 40mL of 8M Urea 1% (w / w) DL-Dithioteitro (DTT) and stopped in a Heidolph Diax 900 homogenizer. The suspension was covered and allowed to stir for 1 hour to solubilize the inclusion bodies. reduce TGF-β isoforms to their denatured monomeric forms. The suspensions were centrifuged at 12,000 for 30 min at 40 ° C. Supernatants were dialyzed to trocars from 8M Urea (biomedical ICN) to 10% (v / v) acetic acid. E.coli proteins that were soluble in 8M urea precipitated out of solution when the buffer was changed to 10% (v / v) .1% (w / v) acetic acid DTT (Sigma) was added to the suspensions , covered and allowed to wean for 30 min to reduce any disulfide bond that may have formed between TGF-β monomers at buffer exchange. The suspensions were centrifuged to separate soluble and non-soluble proteins. Samples were taken from urea solubilization and pad exchange steps (soluble and non-soluble acetic acid material), and then analyzed using SDS-PAGE.

1.8 Ultrafiltration

The 10% (v / v) acetic acid material from section 1.7 underwent ultrafiltration with a 10kDa membrane on a Vivoflow50 (Vivascience). The purpose of this was to reduce the 10% (v / v) acetic acid suspension volume to 3mL and remove low molecular weight proteins.

1.9 Refolding and Purification

Solubilized TGF-B proteins 1, 2 and 3 were first concentrated by ultrafiltration and then performed by an exclusion size chromatography (SEC) column. SEC fractions containing TGF-β protein were then associated and lyophilized. The freeze-dried monomeric TGF-B 1, 2 and 3 proteins were solubilized in 8M urea containing 10mM DTT until a final concentration of 10mg / mL was reached. TGF-B protein solutions 1, 2 and 3 were added dropwise while stirring the refolding solution (1M 3- (pyridine) -1-propane sulfonate (NDSB-201), 20% (v / v) Dimethyl Sulfoxide (DMSO1 Sigma), 2% (w / v) 3- (3-Cholamidopropyl) dimethylammonium-1-propanesulfonate (CHAPS), 1M NaCl (Sigma), 1% (w / v) reduced glutathione (GSH) , Sigma), 0.05M Triz-ma®Base (Sigma) pH 9.3) until final concentrations of 0.2 mg / mL TGF-B 1, 2 and 3 proteins were reached. It is important that the pH be kept within a range of 9.2-9.4 using concentrated NaOH / HCl. The solution was covered with the Parapelicle, which was punctured to allow the monomeric proteins to be oxidized and allowed to stir at 8 ° C. After 144 hours the solution was centrifuged at 15,000g for 40 minutes to remove the precipitate formed and the pH adjusted to pH 3.5 with aceticoglacial acid.

1.10 Purification of Monomeric Refolding Proteins

The resulting refolded TGF-B material was then further purified using standard chromatographic techniques followed by concentration of the relevant fractions (i.e. containing TGF-B monomeric proteins) by ultrafiltration prior to characterization. IN VITRO AND IN VIVO METHODOLOGY FOR CHARACTERIZATION AND EVALUATION OF BIOLOGICAL ACTIVITY OF TGF-B3MONOMIC PROTEINS.

Monomeric TGF-β proteins have been characterized and evaluated for biological activity via several methods including: SDS-PAGE, amino acid sequence analysis (by LCMS); in vivo biological activity assay (using mouse models of skin wound healing and scars described below).

2.1 IN VITRO CHARACTERIZATION OF TGF-B3 MONOMERS.

2.1.1 Reduction and non-reduction SDS-PAGE analysis of monomeric purified TGF-B3

2.1.1.1 Method

Wild-type purified TGF-B3 monomers were evaluated by SDS-PAGE to determine purity and molecular weight. 3pg purified TGF-B3 demonomers (non-reduced reduced samples), positive TGF-B3 3pg (reduced and unreduced) positive control and 10pL Invitrogen Mark 12 molecular weight standards were loaded onto a polyacrylamide gel ( 10% -20% (v / v) acrylamide gradient). Once the electrophoresis was complete the gel was stained with Coomassie Blue.

2.1.1.2 Results

Non-reduction and reduction SDS-PAGE analysis results are shown in Figure 1, and illustrate that dimeric TGF-B3 functions at the expected gel positions (~ 13kDa for reduced as e25kDa monomers for non-reduction samples ). Monomeric TGF-β functionalized at the expected position of ~ 13kDa (theoretical 12.5kDa) on the gel for reduced and non-reduced samples. No additional protein strips have been detected by Coomassie Blue staining, which has a sensitivity of approximately 1pg of protein.

2.1.2 Amino Acid Sequence Analysis

2.1.2.1 Method

50 microliters of purified monomeric TGF-B3 was dried in vacuo and then resuspended in 20μΙ of a solution containing 50mM NH4HCO3 and 10% (v / v) acetonitrile. 20μς of sequenced class Tripsin (Promega) resuspended in 10μΙ of resuspension buffer supplied by Kit (Promega) to give a Trypsin concentration of 2pg / pl. This was then diluted in 50mM NH4HCO3 and 10% (v / v) acetonitrile to give a final trypsin concentration of 0.2pg / W. Digestion was performed overnight by the addition of trypsin in a 1:20 (w / w) ratio of monomeric TGF-β3. Digestion was abruptly cooled by the addition of formic acid (FIuka) to a final concentration of 0.1% (v / v). The samples were then diluted to 1pmol / μΙ and the peptides were then analyzed by an RP-LCMS nanoflow process (last system, Dionex online for a Q-ToF2, Micromass). Chromatography was performed on a 75 pm C18 column (LC packaging) using a 45 min gradient from 5% deacetonitrile to 55% acetonitrile (Romil). MS analysis takes the form of data-dependent analysis where instruments measured m / z of peptide ions eluting from LC and selecting appropriate ions for MSMS analysis where collidally induced decomposition was employed to fragment peptide ions to render information of peptide ions. sequence.

2.1.2.2 Results

Amino acid sequence analysis confirmed that the "wild type" TGF-B3 monomer had the expected amino acid sequence (corresponding to Sequence ID N-3).

2 IN VIVO EVALUATION OF BIOLOGICAL ACTIVITY OF DETGF-B3 MONOMERS.

Literature data have shown that dimeric TGF-Bs, including TGF-B3, accelerate skin healing and reduce scarring followed by injury or injury (Shah, M et al. 1995). The following example investigated the biological effects of monomeric TGF-63 on wound healing (3 days post-wound) and scarring (70 days post-wound) in adult male rats.2.2.1 The Effect of Mutant TGF-B3 Proteins and "wild type" in Wound Healing (Incisional Wound Day 3).

2.2.1.1 Method

Male rats (Sprague Dawley) were anesthetized with Halo-thane and its shaved backs. Wound positions were marked using a standard proprietary model with skin marking ink as shown in Figure 2. Samples were diluted in sterile vehicle buffer containing 0.25M Maltose (Sigma), 0.002% (v / v) acetic acid and 0, 33% (v / v) isopropyl alcohol at concentrations outlined in the table in figure 3.All samples were sterile filtered and endotoxin free. Four rats were used for each treatment group. 100pL of sample from each treatment group (Figure 3) were injected intradermally into the marked A and B wound positions (except rats not receiving any "naive" treatment). In AeB wound positions long 1cm thick incisions were made with an N5 11 scalpel blade. All animals were caged separately. After 24 hours the animals received a second dose of the sample. After 3 days the wounds were photographed and analyzed using a macroscopic Visual Analog Count system (modified from Beausang, E et al 1998). Statistical analysis was performed using Mann Whitney U / Student T tests. A value of p <0.05 was considered significant.

2.2.1.2 Results

Incisional wounds were examined after 3 days using the macroscopic VAS system (see Figure 4). On this ten-point scale an index of 0 represents a well-healed wound and an index of 10 represents a very poorly healed wound. The data show that:

Treatment with 50ng / 100pL or 100ng / 100pL of wild-type mo-nomic TGF-B3 decreases the VAS index (ie, improves macroscopic appearance of wounds) compared to both without treatment (naïve control). ") and placebo treatment. Treatment with 100ng / 100pL dose significantly (p <0.01) improved the appearance of wounds compared to no treatment. It is surprising to note that wild-type monomeric TGF-β showed greater efficiency than the dimeric form.2.2 The Effect of "Wild-Type" Monomeric TGF-B3 Proteins on Scar (Incisional wound day 70).

2.2.2.1 MethodMale Sprague Dawley rats were anesthetized with Halo-thane and its shaved backs. Wound positions were marked using a standard skin marking ink model as shown in Figure 5. Samples were diluted in sterile vehicle buffer containing 0.25M Maltose (Sigma), 0.002% (v / v) acetic acid and 0.33% (v / v) isopropyl alcohol at concentrations outlined in the table in figure 5.

All samples were filter sterilized and free of endotoxin. Four rats were used for each treatment group. 10OpL of the sample from each treatment group (Figure 5) were injected intradermally into the marked A and B wound positions (except rats receiving no treatment - "naive"). In the AeBa wound positions 1 cm long incisions were made with a No. 11 scalpel blade. All animals were caged separately. After 24 hours the animals received a second dose of the sample. After 70 days the scars were photographed and analyzed using a macroscopic Visu-al Analog Counting system (modified from Beausang, E et al 1998). Representative examples of the macroscopic appearance of scars produced as described above are shown in Figure 7.

After macroscopic analysis, the wounds were cut and placed in 10% buffered saline before being processed for histological evaluation in wax blocks. The wax blocks were cut into 5 pm serial sections and placed on slides. The slides were stained with Massons Trichrome and analyzed. Statistical analysis of the data was performed using Mann Whitney U / Student T tests. A p value <0.05 was considered significant. Representative examples of the microscopic appearance of scars were shown in figure 8.

2.2.2.2 Evaluation of Incisional Wounds from Day 70 using Macroscopic VAS

Incisional wounds were examined after 70 days using a macroscopic VAS system. An index of 10 indicates a bad scar and an index of 0 is normal skin. The results of the VAS analysis of day70 wounds are summarized in Figure 6. This shows that: "wild-type" monomeric TGF-33 (at doses of SOng / 100pL and 100ng / 100pL) reduced scarring compared to placebo-treated naive wounds. For the 100ng / 100pL dose this reduction is statistically significant compared to placebo-treated wounds (p <0.01).

Dimeric TGF-B3 in SOng / 100pL reduced scar compared to naive and placebo-treated scars. This reduction is statistically compared to placebo-treated wounds (p <0.01).

3. DEVELOPMENT OF PREFERRED CONDITIONS FOR THE PRODUCTION OF BIOLOGICALLY ACTIVE MONOMERS.

The inventors investigated whether improved methods of generating biologically active, correctly folded TGF-β monomers could not be established. The following example is illustrative of the application of the methods of the invention to monomeric refolding of TGF-B3.

A study has been established to select refolding reagents (see 3.12). Methods of purifying correctly folded monomers were then investigated (3.13).

These studies led the inventors to find that improved methods of biologically generating correctly folded TGF-B3 monomers can be established by following a method comprising solubilized addition, unfolded monomeric growth factor for a solution containing:

(i) 2- (cilcohexylamino) ethanesulfonic acid (CHES) or a functional analog thereof; and

(ii) low molecular weight sulfhydryl / disulfide redox system; and

incubate the growth factor in the solution until the dimeric biologically active growth factor is formed.

EXPERIMENT:

3.1 cDNA Generation

Total RNA from a human incisional wound (taken on day 5 post-wound) was treated with Free DNA (Ambion) to remove any contamination DNA. Using total RNA as a template, tGF-B3 cDNA was generated by Polymerase-Transciptase Reverse (RT-PCR) chain reaction. The master RT-PCR mixture was prepared from the Brilliant® QRT-PCR Core Reactor Kit, 1 step (Stratagene). An RNA microorganism was added to 50μΙ of a solution containing: One Step QRT-PCR Buffer, 0.2mM dNTPs, 3.5mM MgCl2, 1pL Reverse Transcript StrataScript, 2.5 Units Taq Polymerase, 0 , 4μΜsense primer (5 'GAT ATA CCA TGG CTT TGG ACA CCA ATT ACT ACT GC 3'), 0.4μΜ sense initiator (5'-CAG CCG GAT CCG GTC GAC TCA GCT ACATTT ACA AG 3 ') · The reaction It was placed in a thermal cycler (HybaidPCR Express) and functionalized under the following conditions: 30 min at 45 ° C, 10 min at 95 ° C, then 40 cycles of 95 ° C for 30s, 65 ° C for 1 min and 72 ° C for 1min. The final step is 72 ° C for 10 min. PCR samples were functionalized in 2% (w / w) agarose gel to verify strip size and purified using Wizard PCR Prep Kit (Promega).

3.2 Vector Cloning and Host Cell Transformation

The pET-24d vector is derived from the pBR322 vector and contains a T7 promoter under LacUV5 control and a Kanamycin resistant marker gene.

TGF-B3 cDNA fragments (generated in section 1.1) were digested with 0.75μ1_ Ncol (New England Biolabs) and 0.75μΙ_ deBamHI (New England Biolabs) with 1 X BamHI buffer (New England Biolabs). labs) in a reaction of 15μΙ_ (Nuclease Free Water, Novagen) at 37 ° C for 4 hours. One microliter of plasmid pET-24d (Novagen) was digested in the same manner. The digested cDNA and large plasmid fragment were purified gelagarose and recovered using the SpinPrepGel DNA Extraction Kit (Novagen).

Purified plasmid and cDNA fragments were ligated using the T4 Ligase Kit (Novagen). The cDNA / bound plasmid was transformed into HMS174 (DE3) (Novagen HMS174 (DE3) transformation kit).

Transformants were selected by lamination on Luria decal agar (LB) plates containing 50μg / ml Kanamycin (Invitrogen). Three colones were selected for restriction of digestion and / or expression.3.3 Clone Selection for Product Expression

Clones were grown in half-strength "Terrific" shake flask (6g / L phytone petone (Becton Dickin-son), 12g / L yeast extract (Becton Dickinson), 2g / L glycerol (JTBaker) cultures. ), 1.16g / L monobasic potassium phosphate (JT Baker), 6.25g / L dibasic potassium phosphate (JT Baker), QS to 1 liter with distilled water) and exponentially induced in OD6oo between 0.65 and 0.85 with 1mM deisopropyl beta-D-thiogalactopyranide (IPTG). Post-induction samples were taken 3 hours after IPTG addition and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for product induction and expression. Clones 1-4 aliquot pre / post induction sample and Mark 12 molecular weight standards (molecular weight range 2.5-200kDa - Invitogen) were functionalized on NuPAGE @ Novex12% Bis-Tris, 1.0mm gel ( Invitrogen) for approximately 40-50 minutes at 120milliAmps and 200 volts and then stained with Coomassie Blue. A protein whose size is between 6 and 14.4kDa (i.e. TGF-B3 monomer) is clearly induced in each of these cultures (see Figure 9) with clone 2 expressing the large amount of TGF-B3 protein.

3.4 Frozen Cell Stock

Clones 1-4 were grown in shake flasks in medium-strength BrothTerrific to an OD60 of approximately 1 and stored as glycerol stocks by the addition of 20% (v / v) glycerol. 1.2mL broth was dosed in 12 χ 2mL cryovans (containing 0.3mL glycerol) and then stored at -70 ° C.

3.5 TGF-Beta 3 Gene Sequence Confirmation

Samples of the cultures used for frozen cell stocks were taken prior to glycerol addition and used for plasmid isolation using a Qiagen MiniPrep Kit. The isolated plasmid was sequenced and verified using a T7 promoter primer (5'-TAA). TAC GACTCA CTA TAG GG-3 ') and a T7 terminal primer (5'-GCT AGT TAT TGCTCA GCG G-3'). 3.6 Seed Culture

As clone 2 expressed the highest amount of TGF-Beta 3 protein, a frozen stock vial (from section 3.4) was recovered and inoculated into a 2 liter baffled Erlenmeyer flask containing 500mL HySoy medium (12g / ml). Hy-Soy L (Quest International), 24g / L yeast extract (Becton Dickinson), 10g / L NaCl (Sigma), 10g / L deglycerol (Sigma) and 50pg / ml kanamycin. 37 ° C and 200rpm and sampled periodically to measure OD550. When the culture OD reached 3.21 U / mL (after 7 hours) the cell broth was used to seed a 150L fermenter (100 liters working volume). ).

3.7 Fermentation

Nine hundred milliliters of cell broth (from section 3.6) was used to inoculate 150L of fermenter (WHE) containing 90L of Batch Culture Media (0.6g / L K2HPO4, 0.4g / L KH2HPO4, 1.25g / L NH4SO4 , 12g / l HY-Soy, 24g / l yeast extract, 10g / l glycerol). The fermentation operating parameters were controlled as follows: temperature setting point, 37 ° C; pH setpoint, 7.0 (maintained using 4N ammonium hydroxide and 4N phosphoric acid), and; dissolved oxygen (DO) initially calibrated at 100%. The vessel head pressure was 48.19kPa (0.4914 kgf / cm2), and the agitation and air flow were 200-400rpm with a volume volume of air per half minute (vvm or slpm), respectively. . DO was maintained above 20% by adjusting the fermentation setpoint parameters at the following priority: Agitation (max 400rpm), aeration (max 1.5vvm), oxygen supplement (max 33.3 lpm), 82.61 kPa (maximum 0.8424 kgf / cm2). Foam was controlled with Pluronic L-61 (25% v / v). When the culture OD reached 10U / mL a glycerol feed (50% v / v) was started at a flow rate of 45mL / min. When the OD reached 40 U / ml, the cells were induced with the addition of IPTG at a final concentration of 0.2mM.

3.8 Harvest

After 4 hours postinduction, the fermentor was cooled to 10 ° C and the air flow and stirring reduced to 0.3wm and 100rpm respectively. The foam and pH controls were terminated and the back pressure was adjusted to 20.65 kPa (0.2106 kgf / cm2). The culture was harvested by continuous centrifugation with a Westfalia CSA 8 continuous centrifuge at 10 ° C. The centrifuge was operated at 15,000 rpm and a flow rate of 3 liters per minute and harvested cell slurry.

3.9 Cell Lysis and IB Recovery

The fermentation cell paste (from section 3.8) was diluted 1: 5 with Lysis Buffer (6.1 g / l TrizmaBase (Tris), 3.7 g / l ethylenediaminatetraacetic acid (EDTA), 58.44 l NaCl and 10g / l Triton X-100, pH 8.0) was suspended using a manual homogenizer. The resuspending cell paste was passed twice through a high pressure homogenizer (parameters: pressure, 68.84 kPa (0.702 kgf / cm2); flow rate, 450mL / min; and temperature, 15 ° C) . The homogenized cell lysate was then centrifuged (cuvette centrifuge, fixed angle rotor) at 5.000xg per minute at 4 ° C. The supernatant was discharged leaving insoluble TGF-B3 (inclusion bodies). The inclusion body (IB) pellet was resuspended in Wash Buffer (6.1 g / L Tris and 3.72 g / L EDTA, pH 8.0) using a manual centrifuged homogenizer (5,000 x g per 20 minutes at 4 ° C) .3.10 Inclusion Body Solubilization

The pellet from section 3.9 was diluted 1:10 with Solubilization Buffer (6.1 g / L Tris, 15.4 g / L DL-dithiothreitol (DTT) and Urea 360.4g / L, pH 8.0) and resuspended using a manual homogenizer. The suspension was covered and allowed to stir for 60-75 minutes at room temperature to solubilize the inclusion bodies and reduce TGF-33 to its monomeric form. The pH of the resuspended pellet was adjusted to pH 9.4-9.6 with Na-OH / acetic acid before incubation a second time for 60-75 minutes.

3.11 Clarification / Ultrafiltration and Diafiltration

The solubilized material from section 3.10 was clarified, concentrated and diafiltered in a Tangential Flow Filtration (TFF) system (millipo-re). Initial clarification and concentration were achieved with a preconditioned clarification TFF membrane (Millipore Pellicon 10000Da, Regenerated Cellulose, filter V). The clarified TGF-B3 was collected in the permeate. By switching to a UItrafiltration / Diafiltration (UF / DF) membrane (MilliporePellicon 5kDa, Regenerated Cellulose, filter C), the TGF-G3 was then washed in 6 Solubilization Buffer diavolumes (6, 1 g / l Tris, 15.4 g / l DTT and 360.4 g / l urea, pH 9.5) .3.12 Refolding Screen Matrix 13.12.1 Methodology.

TGF-B3 protein content in the clarified material of section 1.12 was quantified using the RC DC ™ Protein Assay (BioRad) together with SDS-PAGE, Coomassie Blue Staining and densitometry. TGF-Beta 3 material was diluted in a series of refolding buffers (see Tables 1 and 2). 1mL samples were taken daily over a 6 day timeline and analyzed under non-reducing conditions using SDS-PAGE. Sample aliquots and molecular weight standards Mark 12 (2.5-200kDa molecular weight range - Invitrogen) were functionalized on NuPAGE® Novex 12% Bis-Tris Gels, 1.0mm (Invitrogen) for approximately 40-50 minutes in 120 milliAmps and 200 volts. The protein samples were then electrophoretically transferred to the denitrocellulose membrane (Invitrogen) using a Novex Absorption apparatus (Invitrogen). The membrane was blocked with 5% (w / v) skim milk powder, 1% (v / v) polyoxyethylene sorbitan monolaurate ( Tween 20; Sigma) in phosphate buffered saline (PBS). The membrane was washed (PBS, 0.1% (v / v) Tween 20) and incubated for 1 hour in primary antibody (anti-TGF-B3, MAB643 (R&D systems) diluted 1: 500 in PBS and 0.1 ( v / v) Tween 20). After incubation with the primary antibody, nitrocellulose was washed wash buffer (PBS, 1% (v / v) Tween 20). The nitrocellulose was then incubated for an additional 1 hour on the secondary antibody (1gG guinea pig conjugated alkaline phosphatase (Abcam P0397) diluted 1: 2000 with PBS, 0.1% (v / v) Tween 20). The membrane was again washed (PBS, 1% (v / v) Tween20 before developing with Western Blue Stabilized Alkaline Phosphate Substrate (Promega). MAB 643 antibody correctly detects monomeric and dimeric TGF-Beta 3 species redoubled.

3.12.2 Results

Tables 1 and 2 summarize the results obtained by testing TGF-B3 folding under 50 different experimental conditions.

The inventors have established that the conditions described below (i.e. experimental conditions 12, 19, 36, 37, 42, 43 and 44) produced correctly refolded dimeric TGF-Beta 3.

1. 0.7M 2-ethanesulfonic acid (cyclohexylamino) (CHES), 2mM reduced glutathione (GSH), 0.4mM oxidized glutathione (GSSG), 0.12mg / ml_ TGF-Beta 3, pH 9.5 to 2-8 ° C (experimental condition 12).

2. 30mM Taurodeoxycholate, 0.7M CHES, 2mM GSH, 0.4mM GSSG, 0.12mg / mL TGF-Beta 3, pH 9.5 at 2-8 ° C (experimental condition 19) .

3. 1M NDSB-201, 2nM reduced glutathione (GSH), 2mM oxidized deglutathione (GSSG), 0.12mg / ml TGF-Beta 3, pH 9.5 at 2-8 ° C (experimental condition 36).

4. 0.7M CHES, 2mM reduced glutathione (GSH), 2nM oxidized deglutathione (GSSG), 0.12mg / ml TGF-Beta 3, pH 9.5 at 2-8 ° C (experimental condition 37).

5. 30mM Taurodeoxycholate plus 1M NDSB-221, 2mM reduced deglutathione (GSH), 2mM oxidized glutathione (GSSG), 0.12mg / mL TGF-Beta 3, pH 9.5 at 2-8 ° C (experimental condition 42).

6. 30mM Taurodeoxycholate plus 0.7M CHES, 2mM reduced glutathione (GSH), 2mM oxidized glutathione (GSSG), 0.12mg / mL TGF-Beta 3, pH 9.5 at 2-8 ° C (experimental condition 43).

7. 30mM Taurodeoxycholate, 0.7M CHES, 2mM GSH, 2mM GSSG, 0.12mg / mL TGF-Beta 3, pH 9.5 at 2-8 ° C (experimental condition 44).

As an example, Figure 10 illustrates the effectiveness of experimental condition 12. <table> table see original document page 63 </column> </row> <table> <table> table see original document page 64 </column> </ row> <table> Table 2.

<table> table see original document page 65 </column> </row> <table> <table> table see original document page 66 </column> </row> <table> 3.13 Hydrophobic Interaction Ultrafiltration / Chromatography

The selected refolding solution was concentrated in 5 folds by ultrafiltration (the membrane was a Milli-pore Pellicon 5kDa, 0.1 m2, Regenerated Cellulose flat-screen sieve). The pH of the concentrated refolding material was then adjusted to a pH of 2.5-2.8 using glacial acetic acid before being diluted 1: 1 in Dilution Buffer (2.72 g / l of acetate). sodium, 264.28g / l ammonium sulfate 100g / l acetic acid, and 210.7g / l arginine hydrochloride pH 3.3). A Butyl Sepharose Fast Flow 4 Column (Amersham, 16cm Bed Heiht) was equilibrated with four column volumes of Buffer A (2.72g / L sodium acetate, 132.14g / L ammonium sulfate and 100g / L of acetic acid pH 3.3). Refolding material was filtered through the 0.22μΜ membrane (Milli-pore Millipak filter) before being loaded onto the Sepharose butyl column at a flow rate of 10Ocm / h (this flow rate was used throughout the process). -diment). The column was then washed in Buffer A for four column volumes. TGF-Beta 3 proteins eluted from the column using Buffer B (2.72g / l sodium acetate, 100g / l acetic acid and 300g / l ethyl alcohol pH 3.3). The first peak, which contains TGF-β 3 proteins in both monomeric and dimeric forms, was associated (see Figure 11) prior to the separation of monomeric proteins. Sequence InformationTGF-B-1 (Sequence ID N9 1)

ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCSTGF-B-2 (Sequence ID Nq)

ALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCSTGF-B-3 (String ID # 3)

ALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLS (shown in sequence) is shown in peptide (normal)

atgccgccct ccgggctgcg gctgctgctg ctgctgctac cgctgctgtg gctactggtg ctgacgcctg gccggccggc cgcgggacta tccacctgca agactatcga catggagctg gtgaagcgga agcgcatcga ggccatccgc ggccagatcc tgtccaagct gcggctcgcc agccccccga gccaggggga ggtgccgccc ggcccgctgc ccgaggccgt gctcgccctg tacaacagca cccgcgaccg ggtggccggg gagagtgcag aaccggagcc cgagcctgag gccgactact acgccaagga ggtcacccgc gtgctaatgg tggaaaccca caacgaaatc tatgacaagt tcaagcagag tacacacagc atatatatgt tcttcaacac atcagagctc cgagaagcgg tacctgaacc cgtgttgctc tcccgggcag agctgcgtct gctgaggctc aagttaaaag tggagcagca cgtggagctg taccagaaat acagcaacaa ttcctggcga tacctcagca accggctgct ggcacccagc gactcgccag agtggttatc ttttgatgtc accggagttg tgcggcagtg gttgagccgt ggaggggaaa ttgagggctt tcgccttagc gcccactgct cctgtgacag cagggataac acactgcaag tggacatcaa cgggttcact accggccgcc gaggtgacct ggccaccatt catggcatga accggccttt cctgcttctc atggccaccc cgctggagag ggcccagcat ctgcaaagct cccggcaccg ccgagccctg gacaccaact attgcttcag ctccacggag aagaactgct gcgtgcggca gctgtacatt gacttc ACMC aggacctcgg ctggaagtgg atccacgagc ccaagggcta ccatgccaac ttctgcctcg ggccctgccc ctacatttgg agcctggaca cgcagtacag caaggtcctg gccctgtaca accagcataa cccgggcgcc tcggcggcgc cgtgctgcgt gccgcaggcg ctggagccgc tgcccathgt gtactacgtg ggccgcaagc ccaaggtgga gcagctgtcc aacatgatcg tgcgctcctg caagtgcagc tga

Complete DNA coding TGF-Beta 2 (Sequence ID N95) showing signal peptide (shown in italics), pro-peptide (shown in bold) as well as active fragment (shown in normal text) ctgtctacct gcagcacact cgatatggac cagttcatgc gcaagaggat cgaggcgaxccgcgggcaga tcctgagcaa gctgaagctc accagtcccc cagaagacta tcctgagcccgaggaagtcc ccccggaggt gatttccatc tacaacagca ccagggactt gctccaggagaaggcgagcc ggagggcggc cgcctgcgag cgcgagagga gcgacgaaga gtactacgccaaggaggttt acaaaataga catgccgccc ttcttcccct ccgaagccat cccgcccactttctacagac cctacttcag aattgttcga tttgacgtct cagcaatgga gaagaatgcttccaatttgg tgaaagcaga gttcagagtc tttcgtttgc agaacccaaa agccagagtgcctgaacaac ggattgagct atatcagatt ctcaagtcca aagatttaac atctccaacccagcgctaca tcgacagcaa agttgtgaaa acaagagcag aaggcgaatg gctctccttcgatgtaactg atgctgttca tgaatggctt caccataaag acaggaacct gggatttaaaataagcttac actgtccctg ctgcactttt gtaccatcta ataattacat catcccaaataaaagtgaag aactagaagc aagatttgca ggtattgatg gcacctccac atataccagtggtgatca g aaactataaa gtccactagg aaaaaaaaca gtgggaagac cccacatctcctgctaatgt tattgccctc ctacagactt gagtcacaac agaccaaccg gcggaagaagcgtgctttgg atgcggccta ttgctttaga aatgtgcagg ataattgctg cctacgtccactttacattg atttcaagag ggatctaggg tggaaatgga tacacgaacc caaagggtacaatgccaact tctgtgctgg agcatgcccg tatttatgga gttcagacac tcagcacagcagggtcctga gcttatataa taccataaat ccagaagcat ctgcttctcc ttgctgcgtgtcccaagatt tagaacctct aaccattctc tactacattg gcaaaacacc caagattgaacagctttcta atatgattgt aaagtcttgc aa aaatgcagct

Complete DNA coding TGF-Beta 3 (Sequence ID Ng6) showing signal peptide (shown in italics), pro-peptide (shown in bold) as well as active fragment (shown in normal text)

atgaagatgc acttgcaaag ggctctggtg gtcctggccc tgctgaactt tgccacggtc 60agcctctctc tgtccacttg caccaccttg gacttcggcc acatcaagaa gaagagggtg 120gaagccatta ggggacagat cttgagcaag ctcaggctca ccagcccccc tgagccaacg 180gtgatgaccc acgtccccta tcaggtcctg gccctttaca acagcacccg ggagctgctg 240gaggagatgc atggggagag ggaggaaggc tgcacccagg aaaacaccga gtcggaatac 300tatgccaaag aaatccataa attcgacatg atccaggggc tggcggagca caacgaactg 360gctgtctgcc ctaaaggaat tacctccaag gttttccgct tcaatgtgtc ctcagtggag 420aaaaatagaa ccaacctatt ccgagcagaa ttccgggtct tgcgggtgcc caaccccagc 480tctaagcgga atgagcagag gatcgagctc ttccagatcc ttcggccaga tgagcacatt 540gccaaacagc gctatatcgg tggcaagaat ctgcccacac ggggcactgc cgagtggctg 600tcctttgatg tcactgacac tgtgcgtgag tggctgttga gaagagagtc caacttaggt 660ctagaaatca gcattcactg tccatgtcac acctttcagc ccaatggaga tatcctggaa 720aacattcacg aggtgatgga aatcaaattc aaaggcgtgg acaatgagga tgaccatggc 780cgtggagatc tggggcgcct caagaagcag aaggatcacc acaaccctca tctaatcctc 840atgatgattc ccccac accg gctcgacaac ccgggccagg ggggtcagag gaagaagcgg 900gctttggaca ccaattactg cttccgcaac ttggaggaga actgctgtgt gcgccccctc 960tacattgact tccgacagga tctgggctgg aagtgggtcc atgaacctaa gggctactat 1020gccaacttct gctcaggccc ttgcccatac ctccgcagtg cagacacaac ccacagcacg 1080gtgctgggac tgtacaacac tctgaaccct gaagcatctg cctcgccttg ctgcgtgccc 1140caggacctgg agcccctgac catcctgtac tatgttggga ggacccccaa agtggagcag 1200ctctccaaca tggtggtgaa gtcttgtaaa tgtagctga

Sequence ID N9 7 - Active wild type human TGF-β DNA decoding fragment

GCT TTG GAC ACC AAT TAC TGC TTC CGC AAC TTG GAG GAG AAC TGCTGT GTG CGC CCC CTC TAC ATT GAC TTC CGA CAG GAT CTG GGC TGGAAG TGG GTC CAT GAA TAC GCA GAC ACA ACC CAC AGC ACGGTG CTG GGA CTG TAC AAC ACT CTG AAC CCT GAA GCA TCT GCC TCGCCT TGC TGC GTG CCC CAG GAC CTG ACC ATC CTG TAC TATGTT GGG AGC ACC CCC AAA GTG GAG CAG TCT TGT AAA TGT AGC

Sense and antisense primers for cDNA generation of TGF-Beta 1. TGF-Beta 2 and TGF-Beta 3:

TGF-Beta 1

Initiator Senso -5'GTT ACA CCA TGG CCC TGG ACA CCA ACT ATT 3'Antisense Starter - 3 'CAG CCG GAT CCG GTC GAC TCA GCT GCACTT 5'

TGF-Beta 2

Initiator Senso -5 'GTT ACA CCA TGG CTT TGG ATG CGG CCT ATTI Antisense Primer -3' CAG CCG GAT CCG GTC GAC TCA GCT

Sense Initiator - 5 "GAT ATA CCA TGG CTT TGG ACA CCA ATT ACT ACT GC 3'Antisense Initiator - 5 'CAG CCG GAT CCG GTC GAC TCA GCT ACTA TTTACA AG 3'. SEQUENCE LISTING

<110> Renovo LimitedFerguson, MarkMellors, PhilLaverty, HughOccleston, NickO'Kane, SharonAtkinson, Emma <120> MEDICINAL PRODUCTS AND PROTEINS <130> P90083PWO <150> GB0604966.2 <151> 2006-03-11 <160> 15

<170> Patent Version 3.3 <210> 1 <211> 112 <212> PRT <213> Homo sapiens <400> 1

Wing Read Asp Thr Asn Tyr Cys Phe Be Being Thr Glu Lys Asn Cys Cys1 5 10 15

go Arg Gln Read Tyr Ile Asp Phe Arg Lys Asp Read Gly Trp Lys Trp

20 25 3O

Ile His Glu Pro Lys Gly Tyr His Wing Asn Phe Cys Leu Gly Pro Cys35 40 45

Pro Tyr Ile Trp Be Leu Asp Thr Gln Tyr Be Lys Val Leu Wing Leu

50 55 60

Tyr Asn Gln His Asn Pro Gly Wing Be Wing Pro Wing Cys Cys Val Pro65 70 75 80

Gln Wing Leu Glu Pro Leu Pro Ile Val Tyr Tyr Val Gly Arg Lys Pro

85 90 95Lys Val Glu Gln Read Asn Met Ile Val Arg Be Cys Lys Cys Ser100 105 110

<210> 2 <211> 112 <212> PRT

<213> Homo sapiens <400> 2

Wing Read Asp Wing Wing Tyr Cys Phe Arg Asn Val Gln Asp Asn Cys Cys1 5 10 15

Read Arg Pro Read Le Tyr Ile Asp Phe Lys Arg Asp Read Gly Trp Lys Trp

20 25 BO

Ile His Glu Pro Lys Gly Tyr Asn Ala Asn Phe Cys Ala Gly Ala Cys

35 40 45

Pro Tyr Leu Trp Being Asp Thr Gln His Being Arg Val Leu Being Leu 50 55 60

Tyr Asn Thr Ile Asn Pro Glu Ala Ser Ala Ser Pro Cys Cys Val Ser65 70 75 80

Gln Asp Leu Glu Pro Leu Thr Ile Leu Tyr Tyr Ile Gly Lys Thr Pro85 90 95

Lys Ile Glu Gln Read Asn Met Ile Val Lys Be Cys Lys Cys Be

100 105 110

<210> 3 <211> 112 <212> PRT <213> Homo sapiens <400> 3

Wing Read Asp Thr Asn Tyr Cys Phe Arg Asn Read Glu Glu Asn Cys Cys1 5 10 15

go Arg Pro Read Tyr Ile Asp Phe Arg Gln Asp Read Gly Trp Lys Trp 20 25 30

Val His Glu Pro Lys Gly Tyr Tyr Wing Asn Phe Cys Ser Gly Pro Cys35 40 45Pro Tyr Leu Arg Ser Wing Asp Thr Thr His Ser Thr Val Leu Gly Leu

50 55 60

Tyr Asn Thr Read Asn Pro Glu Wing Be Wing Be Pro Cys Cys Val Pro

65 70 75 80

Gln Asp Leu Glu Pro Leu Thr Ile Leu Tyr Tyr Val Gly Arg Thr

85 90 95

Lys Val Glu Gln Leu Ser Asn Met Val Val Lys Ser Cys Lys Cys Ser100 105 110

<210> 4 <211> 1173 <212> DNA <213> Homo sapiens <400> 4

atgccgccct ccgggctgcg gctgctgctg ctgctgctac cgctgctgtg gctactggtg 60

ctgacgcctg gccggccggc cgcgggacta tccacctgca agactatcga catggagctg 120

gtgaagcgga agcgcatcga ggccatccgc ggccagatcc tgtccaagct gcggctcgcc 180

agccccccga gccaggggga ggtgccgccc ggcccgctgc ccgaggccgt gctcgccctg 240

tacaacagca cccgcgaccg ggtggccggg gagagtgcag aaccggagcc cgagcctgag 300

gccgactact acgccaagga ggtcacccgc gtgctaatgg tggaaaccca caacgaaatc 360

tatgacaagt tcaagcagag tacacacagc atatatatgt tcttcaacac atcagagctc 420

cgagaagcgg tacctgaacc cgtgttgctc tcccgggcag agctgcgtct gctgaggctc 480

aagttaaaag tggagcagca cgtggagctg taccagaaat acagcaacaa ttcctggcga 540

tacctcagca accggctgct ggcacccagc gactcgccag agtggttatc ttttgatgtc 600

accggagttg tgcggcagtg gttgagccgt ggaggggaaa ttgagggctt tcgccttagc 660

gcccactgct cctgtgacag cagggataac acactgcaag tggacatcaa cgggttcact 720

accggccgcc gaggtgacct ggccaccatt catggcatga accggccttt cctgcttctc 780

atggccaccc cgctggagag ggcccagcat ctgcaaagct cccggcaccg ccgagccctg 840

gacaccaact attgcttcag ctccacggag aagaactgct gcgtgcggca gctgtacatt 900

gacttccgca aggacctcgg ctggaagtgg atccacgagc ccaagggcta ccatgccaac 960

ttctgcctcg ggccctgccc ctacatttgg agcctggaca cgcagtacag caaggtcctg 1020

gccctgtaca accagcataa cccgggcgcc tcggcggcgc cgtgctgcgt gccgcaggcg 1080

ctggagccgc tgcccathgt gtactacgtg ggccgcaagc ccaaggtgga gcagctgtcc 1140aacatgatcg tgcgctcctg caagtgcagc tga 1173

<210> 5 <211> 1182 <212> DNA <213> Homo sapiens <400> 5

ctgtctacct gcagcacact cgatatggac cagttcatgc gcaagaggat cgaggcgatc 60 cgcgggcaga tcctgagcaa gctgaagctc accagtcccc cagaagacta tcctgagccc 120 gaggaagtcc ccccggaggt gatttccatc tacaacagca ccagggactt gctccaggag 180 aaggcgagcc ggagggcggc cgcctgcgag cgcgagagga gcgacgaaga gtactacgcc 240 aaggaggttt acaaaataga catgccgccc ttcttcccct ccgaagccat cccgcccact 300 ttctacagac cctacttcag aattgttcga tttgacgtct cagcaatgga gaagaatgct 360 tccaatttgg tgaaagcaga gttcagagtc tttcgtttgc agaacccaaa agccagagtg 420 cctgaacaac ggattgagct atatcagatt ctcaagtcca aagatttaac atctccaacc 480 cagcgctaca tcgacagcaa agttgtgaaa acaagagcag aaggcgaatg gctctccttc 540 gatgtaactg atgctgttca tgaatggctt caccataaag acaggaacct gggatttaaa 600 ataagcttac actgtccctg ctgcactttt gtaccatcta ataattacat catcccaaat 660 aaaagtgaag aactagaagc aagatttgca ggtattgatg gcacctccac atataccagt 720 ggtgatcaga aaactataaa gtccactagg aaaaaaaaca gtgggaagac cccacatctc 780 ctgctaatgt tattgccctc ctacagactt gagtcacaac agaccaaccg gcggaagaag 840 cgtgctttgg atgcggcct ttgctttaga the aatgtgcagg ataattgctg cctacgtcca 900 ctttacattg atttcaagag ggatctaggg tggaaatgga tacacgaacc caaagggtac 960 aatgccaact tctgtgctgg agcatgcccg tatttatgga gttcagacac tcagcacagc 1020 agggtcctga gcttatataa taccataaat ccagaagcat ctgcttctcc ttgctgcgtg 1080 tcccaagatt tagaacctct aaccattctc tactacattg gcaaaacacc caagattgaa 1140 cagctttcta atatgattgt aaagtcttgc aaatgcagct aa 1182

<210> 6 <211> 1239 <212> DNA <213> Homo sapiens <400> 6

atgaagatgc acttgcaaag ggctctggtg gtcctggccc tgctgaactt tgccacggtc 60agcctctctc tgtccacttg caccaccttg gaagccatta ggggacagat cttgagcaag gtgatgaccc acgtccccta tcaggtcctg gaggagatgc atggggagag ggaggaaggc tatgccaaag aaatccataa attcgacatg gctgtctgcc ctaaaggaat tacctccaag aaaaatagaa ccaacctatt ccgagcagaa tctaagcgga atgagcagag gatcgagctc gccaaacagc gctatatcgg tggcaagaat tcctttgatg tcactgacac tgtgcgtgag ctagaaatca gcattcactg tccatgtcac aacattcacg aggtgatgga aatcaaattc cgtggagatc tggggcgcct caagaagcag atgatgattc ccccacaccg gctcgacaac gctttggaca ccaattactg cttccgcaac tacattgact tccgacagga tctgggctgg gccaacttct gctcaggccc ttgcccatac gtgctgggac tgtacaacac tctgaaccct caggacctgg agcccctgac catcctgtac ctctcgggat

<210> 7 <211> 336 <212> DNA <213> Homo sapiens <400> 7

gctttggaca ccaattactg cttccgcaactacattgact tccgacagga tctgggctgggccaacttct gctcaggccc ttgcccatacgtgctgggac tgtacaaccctcaggacctgg aggccctgtgtctgtctgtcgtgtcgtgtcgtgt

gacttcggcc acatcaagaa gaagagggtg 120ctcaggctca ccagcccccc tgagccaacg 180gccctttaca acagcacccg ggagctgctg 240tgcacccagg aaaacaccga gtcggaatac 300atccaggggc tggcggagca caacgaactg 360gttttccgct tcaatgtgtc ctcagtggag 420ttccgggtct tgcgggtgcc caaccccagc 480ttccagatcc ttcggccagá tgagcacatt 540ctgcccacac ggggcactgc cgagtggctg 600tggctgttga gaagagagtc caacttaggt 660acctttcagc ccaatggaga tatcctggaa 720aaaggcgtgg acaatgagga tgaccatggc 780aaggatcacc acaaccctca tctaatcctc 840ccgggccagg ggggtcagag gaagaagcgg 900ttggaggaga actgctgtgt gcgccccctc 960aagtgggtcc atgaacctaa gggctactat 1020ctccgcagtg cagacacaac ccacagcacg 1080gaagcatctg cctcgccttg ctgcgtgccc 1140tatgttggga ggacccccaa agtggagcag 1200tgtagctga 1239ttggaggaga actgctgtgt gcgccccctc 60aagtgggtcc atgaacctaa gggctactat 120ctccgcagtg cagacacaac ccacagcacg 180gaagcatctg cctcgccttg ctgcgtgccc 240tatgttggga ggacccccaa agtggagcag 300tgtagc 336 <211> 30

<212> DNA

<213> Artificial <220>

<223> Felt initiator for TGF-beta 1

<400> 8

gttacaccat ggccctggac accaactatt 30

<210> 9

<211> 30

<212> DNA

<213> Artificial <220>

<223> Antisense initiator for TGF-beta 1

<400> 9

cagccggatc cggtcgactc agctgcactt 30

<210> 10

<211> 30

<212> DNA

<213> Artificial

<220>

<223> Felt initiator for TGF-beta 2

<400> 10

gttacaccat ggctttggat gcggcctatt 30

<210> 11

<211> 31

<212> DNA

<213> Artificial <220>

<223> Antisense initiator for TGF-beta 2

<400> 11

cagccggatc cggtcgactc agctgcattt g ??? 31

<210> 12 <211> 35

<212> DNA

<213> Artificial <220>

<223> sense primer for TGF-beta 3

<400> 12

gatataccat ggctttggac accaattact actgc 35

<210> 13

<211> 37

<212> DNA

<213> Artificial <220>

<223> Antisense primer for TGF-beta 3

<400> 13

cagccggatc cggtcgactc agctacattt acaagac 37

<210> 14

<211> 20

<212> DNA

<213> Artificial

<220>

<223> T7 Promoter Initiator

<400> 14

taatacgact cactataggg 20

<210> 15

<211> 19

<212> DNA

<213> Artificial <220>

<223> T7 termination initiator

<400> 15

gctagttatt gctcagcgg ??? 19

Claims (23)

1. TGF-β monomer, or a biologically active derivative or fragment thereof, for use as a medicament. 2. TGF-33 monomer, or a biologically active derivative or fragment thereof, for use as a medicament. Use as defined in claim 1 or 2, wherein the TGF-β monomer has a molecular weight of approximately 12kDa. Use as defined in claim 2 or 3, in the preparation of a medicament for use in accelerating wound healing and / or inhibiting scarring. Use as defined in any one of claims 2 to 4, wherein the TGF-B3 monomer, or fragment or derivative thereof, is a wild-type TGF-B3 monomer comprising Sequence ID Ns 3, or a biologically active derivative or fragment of same. Use according to claim 4 or 5, wherein the wound is a dermal wound. Use according to claim 4 or 5, wherein the wound is an eye wound. Use according to any one of claims 4 to 7, wherein the wound is an acute wound. Use according to claim 8, wherein the wound is a burn. Use according to any one of claims 4 to 7, wherein the wound is a chronic wound. Use according to claim 2 or 3, in the preparation of a medicament for use in promoting epithelial regeneration. Use according to claim 11, wherein the epithelium comprises the epidermis. Use according to claim 11, wherein the epithelium comprises an eye epithelium. Use according to claim 2 or 3, in the preparation of a medicament for use in the prevention and / or treatment of a fibrofibrotic disorder. Use according to claim 14, wherein the fi brotic disorder is selected from the group consisting of pulmonary fibrosis, liver fi brosis, scleroderma, skin fibrosis, muscle fibrosis, deradiation fibrosis, kidney fibrosis, eye fibrosis, and uterine fibrosis. Use as defined in any preceding claim, wherein the monomer, or biologically active fragment or derivative thereof, is provided at a concentration between 0.78pM and 0.78mM. Use as defined in any of the preceding claims, wherein the medicament has a pH of approximately 5 to 7. Use as defined in any preceding claim, wherein the medicament is substantially alcohol free. Use as defined in any one of the preceding claims, wherein the TGF-β monomer, or biologically active fragment or derivative thereof, is folded into its biologically active form by a method comprising adding non-folded monomeric TGF-β, solubilized (or fragment or derivative thereof) to a solution containing: (i) 2- (cyclohexylamino) ethanesulfonic acid (CHES) or a functional analogue thereof; and (ii) low molecular weight sulfhydryl / bisulfide redox system, and incubate TGF-β in solution until biologically active dimeric monomeric TGF-β is formed. A method of accelerating wound healing and / or inhibiting scarring, the method comprising administering to a patient in need of treatment a therapeutically effective amount of a monomeric TGF-β or biologically active derivative or fragment thereof. The method of claim 20, the TGF-B3 monomer, or fragment, is a wild-type TGF-B3 monomer comprising Sequence ID N-3, or a biologically active derivative or fragment thereof. The method of claim 20 or 21, wherein said is a dermal wound. The method of claim 20 or 21, wherein said is an eye wound.